Engineered botulinum neurotoxin

ABSTRACT

Disclosed herein is a botulinum neurotoxin (BoNT) polypeptide with a modified receptor binding domain (HC) having one or more amino acid mutations that modify the binding of the BoNT to the receptor. Specific mutations and combinations of mutations are also disclosed. Isolated modified HC, polypeptides comprising the modified HC, chimeric molecules, pharmaceutical compositions, and methods of making and using the same are also disclosed. Methods of identifying additional such modified receptor binding domains, are further disclosed.

RELATED APPLICATIONS

This Application is the National Stage of International Application No.PCT/US16/24211, filed Mar. 25, 2016, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/138,818,filed Mar. 26, 2015, the contents of which are incorporated herein byreference in their entirety.

GOVERNMENTAL SUPPORT

This invention was made with Government support under NIH NCRR RR000168awarded by the National Institute of Health. The Government has certainrights in the invention.

SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), the present specification makesreference to a Sequence Listing (submitted electronically as a .txt filenamed “0342941_0606_SL.TXT”). The .txt file was generated on May 5,2016, and is 95,818 bytes in size. The entire contents of the SequenceListing are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of modified neurotoxins andtheir use for treating medical and/or aesthetic disorders.

BACKGROUND OF THE INVENTION

Botulinum neurotoxins are a family of bacterial toxins, including sevenmajor serotypes (BoNT/A-G)¹. These toxins act by blockingneurotransmitter release from neurons, thus paralyzing animals andhumans. In recent years, BoNTs have been widely used to treat a growinglist of medical conditions: local injections of minute amount of toxinscan attenuate neuronal activity in targeted regions, which can bebeneficial in many medical conditions as well as for cosmeticpurposes²⁻⁴.

BoNT/A and BoNT/B are the only two BoNTs that are currently FDA-approvedfor use in humans²⁻⁴. These are toxins purified from bacteria withoutany sequence modifications (defined as wild type, WT). As theapplication of BoNTs grows, limitations and adverse effects have beenreported. The major limitation is the generation of neutralizingantibodies in patients, which renders future treatment ineffective⁵.Termination of BoNT usage often leaves patients with no other effectiveways to treat/relieve their disorders. The possibility of antibodyresponses is directly related to both toxin doses and the frequency ofinjection⁵. Therefore, this limitation mainly occurs in treating musclespasms, which involves relatively high doses of toxins. Consistently,antibody responses have not been observed in cosmetic applications,which use extremely low toxin doses⁵.

The major adverse effects are also often associated with treating musclespasms, but not cosmetic applications. This is because the adverseeffects are largely due to diffusion of toxins to other regions of thebody and the possibility of toxin diffusion is directly related toinjected doses. The adverse effects ranges from transient non-seriousevents such as ptosis and diplopia to life-threatening events evendeath^(6,7). In a petition letter filed in 2008 by Dr. Sidney Wolfe toFDA, a total of 180 serious adverse events, including 16 deaths havebeen documented. As a result, FDA now requires the “Black box warning”on all BoNT products, highlighting the risk of the spread of toxins,following similar warnings issued by the European Union.

Because both the generation of neutralizing antibodies and toxindiffusion are directly related to injected doses, lowering toxin doses(while maintaining the same levels of toxin activity) is highly desired,which means the efficacy of individual toxin molecules has to beenhanced. Such modified BoNTs with improved specificity for neurons willalso reduce any potential off-target effects due to non-specific entryinto other cell types.

BoNTs target and enter neurons by binding to their specific receptorsthrough their receptor binding domains, which are well-defined in theliterature (BoNT-H_(C), FIG. 1A, B)¹. Receptor binding dictates theefficacy and specificity of BoNTs to recognize neurons. Improving thereceptor binding ability of BoNTs will enhance their efficacy andspecificity to target neurons. The receptors for most BoNTs have beenidentified (FIG. 1C). BoNT/B, D-C, and G share two homologous synapticvesicle proteins synaptotagmin I and II (Syt I/II) as theirreceptors⁸⁻¹³, while BoNT/A, E, D, and F use another synaptic vesicleprotein SV2^(9,14-18). In addition to protein receptors, all BoNTsrequire lipid co-receptor gangliosides (FIG. 1D), which are abundant onneuronal surfaces¹⁹. Among the two Syt isoforms Syt II has ˜10-foldhigher binding affinity for BoNT/B than Syt I and is also the dominantisoform expressed in motor nerve terminals, which are the targetedneurons for BoNTs (FIG. 2A)^(20,21). Therefore, Syt II is considered themajor toxin receptor for BoNT/B, D-C, and G, while Syt I is a minortoxin receptor at motor nerve terminals. This is the case in rodents andlikely in the majority of mammals.

One may argue that BoNTs already have high specificity to neurons, andask is it possible to further improve their binding to neurons? Theanswer is a “Yes” for humans, at least in part in light of the recentdiscovery that the human Syt II has greatly diminished binding andfunction as the receptor for BoNT/B due to a unique amino acid changefrom rodent (rat/mouse) Syt II within the toxin binding site^(13,22).This is a change from phenylalanine (F) to leucine (L) at position 54(mouse Syt II sequence) (FIG. 2B). Sequence alignments have revealedthat phenylalanine at this position is highly conserved in both Syt Iand Syt II across vertebrates, including platypus, fish, rodents, andmonkeys²³. Only human and chimpanzee Syt II contains leucine at thisposition. As a result of this residue change, human and chimpanzee SytII has greatly diminished binding to BoNT/B, D-C, and G (FIG. 2C) and issignificantly less efficient in mediating the entry of BoNT/B (FIG. 2D),as compared to mouse Syt II. Since human and chimpanzee Syt I stillcontains phenylalanine at the same position and can bind BoNT/B, D-C,and G (FIG. 2E), the high affinity receptor for BoNT/B, D-C, and G inhumans is restricted to the minor receptor Syt I. These findings providean explanation for the clinical observations that a much higher dose ofBoNT/B than BoNT/A (which binds a different receptor) is needed toachieve the same levels of therapeutic effects in patients^(24,25).Previously these observations were attributed to other reasons, such asthe percentage of active neurotoxin in the preparations used. The recentobservations of such binding differences of BoNT/B to human Syt IIversus Syt II of other species suggests that different residues ofBoNT/B may be involved in binding to human Syt II. As such, sequencemodification to BoNT/B that is expected to affect binding to rodentSytII may have unpredictable effects on BoNT/B binding to human Syt II.

Thus there is a great need to improve binding of BoNT/B to humanreceptor Syt II, as a way to increase its efficacy and specificity totarget human neurons and decrease the toxin doses needed in therapeuticapplications.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide comprising a protease domain, a protease cleavage site, atranslocation domain, and a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)), comprising one or moresubstitution mutations corresponding to substitution mutations inserotype B, strain 1, selected from the group consisting of E1191C,E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A,W1178S, Y1183C, Y1183P, and combinations thereof. In one embodiment, themodified (B-H_(c)) comprises one substitution mutation corresponding toa substitution mutation in serotype B, strain 1, selected from the groupconsisting of E1191C, E1191V, E1191L, E1191Y, S1199E, S1199H, W1178Y,W1178Q, W1178A, W1178S, Y1183C, and Y1183P. In one embodiment of thepolypeptides described herein the modified (B-H_(c)) comprises thesubstitution mutation corresponding to E1191C in serotype B, strain 1.In one embodiment of the polypeptides described herein the modified(B-H_(c)) comprises the substitution mutation corresponding to E1191V inserotype B, strain 1. In one embodiment of the polypeptides describedherein the modified (B-H_(c)) comprises two substitution mutations.

Another aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide comprising a protease domain, a protease cleavage site, atranslocation domain, and a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)), comprising two or moresubstitution mutations corresponding to substitution mutations inserotype B, strain 1, wherein one of the substitution mutations isselected from the group consisting of E1191Q, E1191M, E1191C, E1191V,E1191L, and E1191Y. In one embodiment, one other of the substitutionmutations corresponds to S1199W, S1199E, S1199H, S1199Y, W1178Y, W1178Q,W1178A, W1178S, Y1183C, Y1183P, S1199F or S1199L in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191M and S1199W, E1191M andW1178Q, E1191C and S1199W; E1191C and S1199Y, E1191C and W1178Q, E1191Qand S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q,in serotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191M and S1199W inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191M and W1178Q inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191C and S1199W inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191C and S1199Y inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191C and W1178Q inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191Q and S1199W inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191V and S1199W inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191V and S1199Y inserotype B, strain 1. In one embodiment of the polypeptides describedherein the two substitution mutations correspond to E1191V and W1178Q inserotype B, strain 1. In one embodiment of the polypeptides describedherein the modified (B-H_(c)) comprises three substitution mutations. Inone embodiment of the polypeptides described herein the threesubstitution mutations are at positions that correspond to E1191, Y1183and S1199 or to E1191, S1199 and W1178 of serotype B, strain 1. In oneembodiment of the polypeptides described herein the three substitutionmutations correspond to E1191M, S1199W and W1178Q of serotype B, strain1.

Another aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide comprising a protease domain, a protease cleavage site, atranslocation domain, and a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)), comprising a substitutionmutation at a position corresponding to S1199 or S1201 of serotype B,strain 1.

In one embodiment of any of the polypeptides described herein themodified B-H_(c) is of strain 1. In one embodiment of any of thepolypeptides described herein the protease domain, translocation domain,and protease cleavage site are from serotype selected from the groupconsisting of A, B, C, D, E, F, G, and combinations thereof. In oneembodiment of any of the polypeptides described herein the proteasedomain, translocation domain, and protease cleavage site are fromserotype B, strain 1. In one embodiment of any of the polypeptidesdescribed herein the protease domain, translocation domain, and proteasecleavage site are from serotype A, strain 1. In one embodiment of any ofthe polypeptides described herein, the modified B-Hc is not of strainB4.

Another aspect of the invention relates to a polypeptide comprising amodified receptor binding domain of Clostridial botulinum serotype B(B-H_(c)) comprising one or more substitution mutations corresponding tosubstitution mutations in serotype B, strain 1, selected from the groupconsisting of E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H,W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinationsthereof. In one embodiment, the modified (B-H_(c)) comprises twosubstitution mutations.

Another aspect of the invention relates to a polypeptide comprising amodified receptor binding domain of Clostridial botulinum serotype B(B-H_(c)) comprising two or more substitution mutations corresponding tosubstitution mutations in serotype B, strain 1, wherein one of thesubstitution mutations is selected from the group consisting of E1191Q,E1191M, E1191C, E1191V, E1191L, and E1191Y. In one embodiment of thepolypeptides described herein one of the substitution mutationscorresponds to S1199W, S1199E, S1199H, S1199Y, W1178Y, W1178Q, W1178A,W1178S, Y1183C, Y1183P, S1199F or S1199L in serotype B, strain 1. In oneembodiment of the polypeptides described herein the two substitutionmutations correspond to E1191M and S1199W, E1191M and W1178Q, E1191C andS1199W; E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191Vand S1199W, E1191V and S1199Y, or E1191V and W1178Q, in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191M and S1199W in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191M and W1178Q in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191C and S1199W in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191C and S1199Y in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191C and W1178Q in serotype B,strain. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191Q and S1199W in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191V and S1199W in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191V and S1199Y in serotype B,strain 1. In one embodiment of the polypeptides described herein the twosubstitution mutations correspond to E1191V and W1178Q in serotype B,strain 1. In one embodiment of the polypeptides described herein themodified (B-H_(c)) comprises three substitution mutations. In oneembodiment of the polypeptides described herein the three substitutionmutations are at positions that correspond to E1191, Y1183 and S1199 orto E1191, S1199 and W1178 of serotype B, strain 1. In one embodiment ofthe polypeptides described herein the three substitution mutationscorrespond to E1191M, S1199W and W1178Q of serotype B, strain 1. In oneembodiment of the polypeptides described herein the modified B-H_(c) isof strain 1. In one embodiment of the polypeptides described herein themodified B-Hc is not of strain 4. In one embodiment, the modified B-Hcis not of strain 3, 7 or 8.

Another aspect of the invention relates to a chimeric moleculecomprising a first portion that is a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)) linked to a second portion,wherein the modified B-H_(c) comprises one or more substitutionmutations selected from the group consisting of E1191C, E1191V, E1191L,E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C,Y1183P, and combinations thereof. In one embodiment the modified B-H_(c)comprises two substitution mutations.

Another aspect of the invention relates to a chimeric moleculecomprising a first portion that is a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)) linked to a second portion,wherein the modified B-H_(c) comprises two or more substitutionmutations corresponding to substitution mutations in serotype B, strain1, wherein one of the substitution mutations is selected from the groupconsisting of E1191Q, E1191M, E1191C, E1191V, E1191L, and E1191Y. In oneembodiment of the chimeric molecules described herein one of thesubstitution mutations corresponds to S1199W, S1199E, S1199H, S1199Y,W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, S1199F or S1199L inserotype B, strain 1. In one embodiment of the chimeric moleculesdescribed herein the two substitution mutations correspond to E1191M andS1199W, E1191M and W1178Q, E1191C and S1199W; E1191C and S1199Y, E1191Cand W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, orE1191V and W1178Q, in serotype B, strain 1. In one embodiment of thechimeric molecules described herein the two substitution mutationscorrespond to E1191M and S1199W in serotype B, strain 1. In oneembodiment of the chimeric molecules described herein the twosubstitution mutations correspond to E1191M and W1178Q in serotype B,strain 1. In one embodiment of the chimeric molecules described hereinthe two substitution mutations correspond to E1191C and S1199W inserotype B, strain 1. In one embodiment of the chimeric moleculesdescribed herein the two substitution mutations correspond to E1191C andS1199Y in serotype B, strain 1. In one embodiment of the chimericmolecules described herein the two substitution mutations correspond toE1191C and W1178Q in serotype B, strain 1. In one embodiment of thechimeric molecules described herein the two substitution mutationscorrespond to E1191Q and S1199W in serotype B, strain 1. In oneembodiment of the chimeric molecules described herein the twosubstitution mutations correspond to E1191V and S1199W in serotype B,strain 1. In one embodiment of the chimeric molecules described hereinthe two substitution mutations correspond to E1191V and S1199Y inserotype B, strain 1. In one embodiment of the chimeric moleculesdescribed herein the two substitution mutations correspond to E1191V andW1178Q in serotype B, strain 1. In one embodiment of the chimericmolecules described herein the modified (B-H_(c)) comprises threesubstitution mutations. In one embodiment of the chimeric moleculesdescribed herein the three substitution mutations are at positions thatcorrespond to E1191, Y1183 and S1199 or to E1191, S1199 and W1178 ofserotype B, strain 1. In one embodiment of the chimeric moleculesdescribed herein the three substitution mutations correspond to E1191M,S1199W and W1178Q of serotype B, strain 1.

In one embodiment of any of the chimeric molecules described herein themodified B-H_(c) is of strain 1. In one embodiment of any of thechimeric molecules described herein, the modified B-Hc is not of strain4. In one embodiment, the modified B-Hc is not of strain 3, 7 or 8.

In one embodiment of any of the chimeric molecules described herein thefirst portion and the second portion are linked covalently. In oneembodiment of any of the chimeric molecules described herein the firstportion and the second portion are linked non-covalently. In oneembodiment of any of the chimeric molecules described herein the secondportion is selected from the group consisting of a small molecule, anucleic acid, a short polypeptide and a protein. In one embodiment ofany of the chimeric molecules described herein the second portion is abioactive molecule. In one embodiment of any of the chimeric moleculesdescribed herein the second portion is a therapeutic polypeptide ornon-polypeptide drug.

Aspects of the invention further relate to any of the BoNT polypeptides,polypeptides or chimeric molecules described herein that exhibitssignificantly enhanced binding of the modified B-Hc to human SytIIand/or significantly reduced binding of the modified B-Hc to human Syt Ias compared to an identical molecule lacking the substitutionmutation(s).

Aspects of the invention further relate to any of the BoNT polypeptides,polypeptides or chimeric molecules described herein wherein thesubstitution mutation produces significantly enhanced binding to humanSytII and/or significantly enhanced binding to human Syt I as comparedto an identical molecule lacking the substitution mutation(s).

Another aspect of the invention relates to a nucleic acid comprising anucleotide sequence that encodes the BoNT polypeptide, polypeptide orchimeric molecule described herein. Another aspect of the inventionrelates to a nucleic acid vector comprising the nucleic acid comprisinga nucleotide sequence that encodes the BoNT polypeptide, polypeptide orchimeric molecule described herein. Another aspect of the inventionrelates to a cell comprising the nucleic acid comprising a nucleotidesequence that encodes the BoNT polypeptide, polypeptide or chimericmolecule described herein, or the nucleic acid vector comprising thenucleic acid.

Another aspect of the invention relates to a cell expressing any of theBoNT polypeptides, polypeptides or chimeric molecules described herein.

Another aspect of the invention relates to a pharmaceutical compositioncomprising any of the botulinum neurotoxin (BoNT) polypeptides describedherein.

Another aspect of the invention relates to a pharmaceutical compositioncomprising any of the polypeptides described herein.

Another aspect of the invention relates to a pharmaceutical compositioncomprising any of the chimeric molecules described herein.

Another aspect of the invention relates to a pharmaceutical compositioncomprising any of the nucleic acids or nucleic acid vectors describedherein.

In one embodiment of any of the pharmaceutical compositions describedherein the pharmaceutical composition further comprises apharmaceutically acceptable excipient.

Another aspect of the invention relates to a kit comprising any of thepharmaceutical compositions described herein and directions fortherapeutic administration of the pharmaceutical composition.

Another aspect of the invention relates to a method to produce any ofthe botulinum neurotoxin (BoNT) polypeptides described herein, themethod comprising the steps of culturing the cell that expresses theBoNT polypeptide under conditions wherein said BoNT polypeptide isproduced. In one embodiment, the method further comprises one or more ofthe steps of recovering the BoNT polypeptide from the culture, purifyingthe BoNT polypeptide, activating the BoNT polypeptide, and/orformulating the BoNT polypeptide.

Another aspect of the invention relates to a method for treating acondition associated with unwanted neuronal activity comprisingadministering a therapeutically effective amount of the BoNT polypeptidedescribed herein to a subject to thereby contact one or more neurons'exhibiting unwanted neuronal activity, to thereby treat the condition.In one embodiment, the condition is selected from the group consistingof, spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia,oromandibular dysphonia, lingual dystonia, cervical dystonia, focal handdystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder,cerebral palsy, focal spasticity and other voice disorders, spasmodiccolitis, neurogenic bladder (i.e. all diseases involving urinaryincontinence, such as e.g. neurogenic detrusor overactivity oridiopathic overactive bladder), anismus, limb spasticity, tics, tremors,bruxism, anal fissure, achalasia, dysphagia and other muscle tonedisorders and other disorders characterized by involuntary movements ofmuscle groups, lacrimation, hyperhydrosis, excessive salivation,excessive gastrointestinal secretions, secretory disorders, pain frommuscle spasms, neuropathic pain, inflammatory pain, headache pain (suchas e.g., migraine), itch (pruritis), acne, and dermatological oraesthetic/cosmetic conditions.

Another aspect of the invention relates to any of the botulinumneurotoxin (BoNT) polypeptides or the pharmaceutical compositions, orthe chimeric molecules, or the polypeptides described herein for use inmedicine.

Another aspect of the invention relates to any of the botulinumneurotoxin (BoNT) polypeptides, or the pharmaceutical compositions, orthe chimeric molecules, or the polypeptides described herein, for use intreating a condition associated with unwanted neuronal activity.

Another aspect of the invention relates to a method for identifying amodified receptor binding domain of botulinum neurotoxin for binding toa receptor comprising expressing the modified receptor binding domain asa first fusion protein with T18 subunit of bacterial adenylate cyclasein a 2-hybrid assay, and expressing the receptor as a second fusionprotein with T25 subunit of bacterial adenylate cyclase in the 2-hybridassay, analyzing a clonal E. coli colony that expresses both the firstand second fusion protein for the presence of a positive indicationabove a negative control, and identifying the modified receptor bindingdomain expressed by the colony exhibiting the positive indication asbinding to the receptor. In one embodiment, the positive indication iscolor development. In one embodiment of the methods described herein,the method further comprises the step of analyzing the colony for thepositive indication against a weakly positive control, and furtheridentifying the modified receptor binding domain expressed by the colonyexhibiting the positive indication above the weakly positive control asbinding to the receptor with high affinity. In one embodiment of themethods described herein, the method is performed with a library ofmodified receptor binding domains each expressed within respectivecolonies. In one embodiment of the methods described herein, thereceptor is human. In one embodiment of the methods described herein,the modified receptor binding domain comprises one or more substitutionmutations, wherein one of the substitution mutations corresponds to amutation in serotype B, strain 1, selected from the group consisting of:E1191M, E1191 Q, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H,S1199F, S1199L W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P andS1199Y.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1D (published data for background) shows schematic modelsfor how BoNTs target neurons (FIG. 1A), their overall protein structure(FIG. 1B), a list of identified receptors (FIG. 1C), and the structuralmodel for BoNT/B binding to its receptors Syt and gangliosides (FIG.1D). (FIG. 1A) A schematic view of BoNT actions: BoNTs recognize neuronsby binding to their specific receptors (step 1), enter neurons viareceptor-mediated endocytosis (step 2), the light chains of BoNTs thentranslocate across endosomal membranes into the cytosol (step 3), wherethese light chains act as proteases to cleave target host proteins (step4). FIG. 1A is adapted from Arnon, S. et al, JAMA, 285:1059, 2001²⁴.(FIG. 1B) BoNTs are composed of a light chain and a heavy chain,connected via a disulfide bond. The heavy chain can be further dividedinto two domains: the translocation domain (H_(N)) and the receptorbinding domain (H_(C)). These functional domains are switchable betweendifferent BoNTs. For instance, BoNT/B-H_(C) can be used to replaceBoNT/A-H_(C) to generate chimeric toxins. (FIG. 1C) A list of identifiedtoxins receptors. (FIG. 1D) A structural model showing binding of BoNT/Bto its protein receptor, Syt (I/II), as well as its lipid co-receptor,gangliosides, on the cell surface. FIG. 1D is adapted from Chai et al,Nature, 444:1096, 2006³¹.

FIG. 2A-FIG. 2D show prior data adapted from published literaturesshowing indicating that human Syt II is not an effective receptor forBoNT/B, D-C, and G. (A) Human Syt II differs from mouse/rat Syt II by asingle residue within the toxin binding site (residue 54 in mouse SytII, 51 in human Syt II). (SytII (mouse) shown is SEQ ID NO: 1 and Syt II(human) shown is SEQ ID NO: 14). (FIG. 2B) Glutathione S-transferase(GST) tagged recombinant mouse Syt II 1-87 (m-Syt II) and a mouse Syt II1-87 mutant mimicking human Syt II (F54L, here and thereafter designedas h-Syt II to simplify the text) were immobilized onglutathione-sepharose beads, and were used to pull down BoNT/B,BoNT/D-C, or BoNT/G, with or without the presence of ganglioside(Gangl). All three toxins bind to m-Syt II 1-87, but not h-Syt II in thepull-down assays. (FIG. 2C) Cultured rat hippocampal neurons onlyexpress Syt I but not Syt II⁸. Therefore, knocking down (KD) Syt Igenerates neurons with no endogenous toxin receptors. Full-length m-SytII and h-Syt II were then expressed in Syt I KD hippocampal neurons, andthese neurons were exposed to BoNT/B (20 nM, 5 min exposure, 24 hoursincubation) or BoNT/D-C (0.3 nM, 5 min exposure, 6 hours incubation) orBoNT/G (40 nM, 5 min exposure, 24 hours incubation). h-Syt II was foundsignificantly less efficient in mediating the entry of BoNT/B, BoNT/D-C,and BoNT/G into Syt I KD neurons as compared to wild type mouse Syt II,as evidenced by the degrees of cleavage of toxin substrate synaptobrevin(Syb). (FIG. 2D) Rat Syt I 1-83 and human Syt I 1-80 were used to pulldown BoNT/B, BoNT/D-C, and BoNT/G, as described in panel C. Human Syt Imediated similar levels of toxin binding as rat Syt I did for all threetoxins. This figure is adapted from our recent publication: Peng et al,J. Cell Science, 2012¹³.

FIG. 3A-FIG. 3B shows residues located in the Syt II binding interfaceon BoNT/B-H_(C), which are candidates for site-directed mutagenesis tomodify the binding affinity. (FIG. 3A) A Close-up view of bindinginterface between BoNT/B-H_(C) and mouse Syt II is shown here. Residuesin BoNT/B-H_(C) that contribute to the binding are marked in bold,whereas residues in Syt II are not bold. Residue F54 in Syt II, in themiddle and shown in slightly larger font size, changes to residue L inhuman Syt II. (FIG. 3B) A list of 19 key residues in BoNT/B that formthe binding pocket of the Syt II binding domain in BoNT/B. Theseresidues are candidates for site-directed mutagenesis.

FIG. 4A-FIG. 4B are illustrations of the bacterial adenylate cyclase twohybrids (BACTH) system that is used to screen BoNT/B-H_(C) mutants fortheir ability to bind human Syt II. (FIG. 4A) A pool of BoNT/B-H_(C)mutants that covers all 20 different amino acids at the selectedposition is generated by PCR amplification with primers containingrandom tri-nucleotides (NNN) at the selected site. A total of 19distinct pools were generated for all 19 residues selected in FIG. 3B.(FIG. 4B) A schematic illustration of the BACTH system. Briefly, thebacterial adenylate cyclase is split to two domains, designated as T25and T18. T25 is fused to human Syt II (residues 1-87) that contains thetoxin binding site, whereas T18 is fused to BoNT/B-H_(C) generated asdescribed in FIG. 4A. These two fusion proteins are encoded on twoseparated plasmids and co-transformed into the same bacteria. Binding ofmutated BoNT/B-H_(C) to human Syt II brings together T25 and T18 domainsin bacteria, which restores the activity of the bacterial adenylatecyclase. This leads to production of cAMP in bacteria, which activatesCAP protein and triggers expression of the reporter gene lacZ thatencodes β-galactosidase. The activity of β-galactosidase leads to bluecolonies on X-gal plates that can be easily identified. Plasmids thatencode BoNT/B-H_(C) can be then extracted from blue colonies andsequenced to identify the specific mutations in BoNT/B-H_(C).

FIG. 5A-FIG. 5B are tables that summarize the results from the BACTHscreening of BoNT/B-H_(C) mutants. (FIG. 5A) List of total colonies andthe number of blue colonies for each pool of BoNT/B-H_(C) mutants fusedto T18, co-expressed in bacteria with human Syt II fused to T25. Basedon Clark-Carbon Equation, the number of total colonies needs to belarger than 380 to achieve a 99.8% of possibility of covering all 20possible amino acids at a single position. Four positions resulted insignificant numbers of blue colonies (68 hours after plating). (FIG. 5B)Plasmids from blue colonies identified in FIG. 5A were extracted andsequenced to determine the specific residue introduced at the indicatedposition in BoNT/B-H_(C). The identified residues are listed. Of note,the blue colonies were further divided to “deep blue colonies”, whichwere found with the position 1191 of BoNT/B-H_(C), and “light bluecolonies”, which were found with the positions 1183, 1199, and 1178. Theterm “deep blue colonies” versus “light blue colonies” were artificiallyjudged by the experimenter based on the differences between the bluecolor of colonies.

FIG. 6A-FIG. 6B shows experimental results that indicate thesemi-quantitative measurement of the interactions between indicatedBoNT/B-H_(C) mutants with human Syt II in BACTH assay. (FIG. 6A) Theβ-galactosidase activity was measured from lysates of bacteria thatexpress both human Syt II fused with T25 and indicated BoNT/B-H_(C)mutants fused with T18. The activity of β-galactosidase presumablyreflects the strength of interactions between human Syt II andBoNT/B-H_(C). WT BoNT/B-H_(C) served as a control. Exchanging residuesE1191 to M, C, V, or Q gave the strongest expression of β-galactosidase.Exchanging residues E1191 to S/A/T/N, Y1183 to C/P, S1199 to W/E/Y/H,W1178 to Y/Q/S showed slight increases of interactions. N=6samples/group. (FIG. 6B) Binding of indicated BoNT/B-H_(C) mutants toeither mouse Syt II (m-Syt II) or human Syt II (h-Syt II), the bindingssites of which are shown in FIG. 2A, was assayed by pull down assay.Exchanging residues E1191 to M, C, V, or Q also showed the strongestbinding to h-Syt II.

FIG. 7A-FIG. 7B shows experimental results of the screening forsecondary mutations in addition to position E1191 that can enhancebinding to h-Syt II. (FIG. 7A) Double mutations were created bycombining E1191M with all other ten residue changes identified in FIG.5B at positions S1199/Y1183/W1178, respectively. These double mutationswere assayed for their ability to bind h-Syt II by pull down assay.Mutants that show strong binding to h-Syt II are indicated with an *(FIG. 7B). Proposed mutations for combination that may enhance theaffinity between BoNT/B and human Syt II are listed in the table.

FIG. 8A-FIG. 8B show the quantitative measurement of binding affinitybetween indicated BoNT/B-H_(C) and h-Syt II. (FIG. 8A) Binding of GSTtagged h-Syt II to His-tagged WT BoNT/B-H_(C) and E1191M/S1199Y wasmeasured by biolayer interferometry assay. Briefly, anti-GST biosensorswere loaded with GST-h-Syt II. The loaded biosensors were incubated with1 μM BoNT/B-H_(C) proteins for measurement of association kinetics,followed by washing steps to measure dissociation kinetics.Representative association and dissociation curves are shown. (FIG. 8B)Binding affinities between indicated BoNT/B-H_(C) and Syt II weremeasured by biolayer interferometry assay as described in panel A.Kinetic parameters (k_(on) and k_(off)) and overall binding affinities(K_(D)) were calculated from a non-linear fit of association anddissociation curves. Binding of WT BoNT/B-H_(C) to h-Syt II is too weakto be reliably determined, with estimated K_(D) beyond the detectionlimit (>20 whereas majority of double and triple mutants showeddrastically increased binding affinity at the range of 0.59 to 4.30 μM.

FIG. 9A-FIG. 9C show experimental results that indicate selectedBoNT/B-H_(C) mutants have enhanced binding to human Syt I. (FIG. 9A) WTBoNT/B-H_(C) and E1191M mutant were purified as His6-tagged recombinantproteins (“His6” disclosed as SEQ ID NO: 13) and incubated withimmobilized GST-tagged h-Syt I (1-80), with or without the presence ofco-receptor gangliosides (Gangl). Binding of WT BoNT/B-H_(C) to h-Syt Irequires the presence of the co-receptor gangliosides, indicating arelatively weak interactions between WT BoNT/B-H_(C) and h-Syt. TheE1191M mutant can bind to h-Syt I without gangliosides, showing improvedbinding affinity to h-Syt I. (FIG. 9B) Binding of WT and indicatedmutant BoNT/B-H_(C) to h-Syt I were measured by biolayer interferometryas described in FIG. 8A. (FIG. 9C) Kinetic parameters (k_(on) andk_(off)) and overall affinities (K_(D)) for WT BoNT/B-H_(C),BoNT/B-H_(C) (E1191M/S1199Y) (“B H_(C) MY”), and BoNT/B-H_(C)(E1191V/S1199Y) (“B H_(C) VY”) to h-Syt I were measured and calculatedas described in FIG. 8B. Binding of WT BoNT/B-H_(C) (B H_(C) wt) toh-Syt I cannot be reliably determined, with estimated KD over thedetection limit (>20 μM). BoNT/B-H_(C) (E1191M/S1199Y, B H_(C), MY) andBoNT/B-H_(C) (E1191V/S1199Y, B H_(C) VY) showed drastically increasedbinding affinity, with K_(D) at 2.9 μM and 5.82 μM, respectively.

FIG. 10A-FIG. 10B are photographs of in situ immunostaining analysisindicating H_(C)B_(MY) showed a robust binding to humanized neurons thatexpress h-Syt II. (FIG. 10A) Humanized neurons were created by knockingdown endogenous Syt I and expressing full-length h-Syt II in culturedrat cortical neurons. Neurons that express full-length m-Syt II or m-SytII (F54L) served as additional controls. These neurons were exposed toWT H_(C)B, followed by immunostaining analysis. Bound H_(C)B wasdetected via a HA tag fused to H_(C)B. Synapsin was labeled as a markerfor presynaptic terminals. WT H_(C)B bound to synapses of WT neurons butnot Syt I KD neurons. Expression of full-length m-Syt II via lentiviraltransduction restored binding of H_(C)B, while expression of full-lengthm-Syt II (F54L) or full-length h-Syt II failed to rescue the binding.(FIG. 10B) Syt I KD also abolished binding of H_(C)B_(MY) to neurons.The binding was rescued by expression of full length m-Syt II, m-Syt II(F54L), and h-Syt II.

FIG. 11A-FIG. 11C are experimental results that indicate BoNT/B_(MY)displayed enhanced efficacy on blocking neurotransmission in humanizedneurons. (FIG. 11A) Humanized neurons were created as described in FIG.11A. These neurons were exposed to a gradient of concentrations offull-length WT BoNT/B or BoNT/B_(MY) for 24 hours. Cell lysates wereharvested and subjected to immunoblotting analysis. β-tubulin served asan internal loading control. More VAMP2 was cleaved by BoNT/B_(MY) thanWT BoNT/B at the same concentrations, indicating that BoNT/B_(MY)entered neurons more efficiently than WT BoNT/B. Humanized neurons wereexposed to a gradient of concentrations of WT BoNT/B or BoNT/B_(MY) for24 hours and then the mIPSC activity was recorded by whole-cellpatch-clamp approach. (FIG. 11B) shows representative mIPSC recordingsat 30 pM toxins. (FIG. 11C) depicts the mIPSC activities versus toxinconcentrations, normalized to neurons that were not exposed to anytoxins. The half maximum inhibitory concentration (IC₅₀) is 89 pM for WTBoNT/B and 7.8 pM for BoNT/B_(MY), demonstrating that enhanced bindingto human receptors resulted in increased efficacy of toxin at functionallevels in neurons.

FIG. 12 is the amino acid sequence of the BoNT/B-Hc (strain 1; BoNT/B1Okra strain). Residues 857-1291 of BoNT/B, strain 1, GenBank:AB232927.1. (SEQ ID NO: 2).

FIG. 13 is the nucleic acid sequence encoding BoNT/B-Hc (strain B1, Okrastrain) residues 857-1291 of BoNT/B, strain 1, based on GenBank:AB232927.1), which has been optimized for expression in E. coli. (SEQ IDNO: 3)

FIG. 14 shows the amino acid sequence of C. botulinum serotype A (1296a.a.). (SEQ ID NO: 4)

FIG. 15 shows the amino acid sequence of C. botulinum serotype B (1291a.a.). (SEQ ID NO: 5)

FIG. 16 shows the amino acid sequence of C. botulinum serotype C1 (1291a.a.). (SEQ ID NO: 6)

FIG. 17 shows the amino acid sequence of C. botulinum serotype D (1276a.a.). (SEQ ID NO: 7)

FIG. 18 shows the amino acid sequence of C. botulinum serotype E (1252a.a.). (SEQ ID NO: 8)

FIG. 19 shows the amino acid sequence of C. botulinum serotype F (1274a.a.). (SEQ ID NO: 9)

FIG. 20 shows the amino acid sequence of C. botulinum serotype G (1297a.a.). (SEQ ID NO: 10)

FIG. 21 shows the amino acid sequence of C. botulinum serotype B, StrainEklund 17B (BoNT/B4) (SEQ ID NO: 11) Genbank Ref: EF051570.1.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to the generation of polypeptides, andnucleic acids encoding the polypeptides, comprising a modified receptorbinding domain of Clostridial botulinum (e.g., serotype B (B-H_(c))),and more particularly to C. botulinum neurotoxin (BoNT) polypeptidewhich has improved binding to its human receptors through theincorporation of a modified receptor binding domain, and nucleic acidsencoding the polypeptide. From these findings, a new generation oftherapeutic BoNTs can be created by utilizing the modified receptorbinding domain identified herein, with improved efficacy and specificityto target human neurons than the currently utilized WT BoNTs.

Definitions

As used herein, the term “binding affinity” means how strong amolecule's binding activity is for a particular receptor system. Ingeneral, high binding affinity results from greater intermolecular forcebetween a binding domain and its receptor system while low bindingaffinity involves less intermolecular force between the ligand and itsreceptor. High binding affinity involves a longer residence time for thebinding domain at its receptor binding site than is the case for lowbinding affinity. As such, a molecule with a high binding affinity meansa lower concentration of that molecule is required to maximally occupythe binding sites of a receptor system and trigger a physiologicalresponse. Conversely, low binding affinity means a relatively highconcentration of a molecule is required before the receptor bindingsites of a receptor system is maximally occupied and the maximumphysiological response is achieved. Thus, a botulinum neurotoxin of thepresent invention with increased binding activity due to high bindingaffinity will allow administration of reduced doses of the toxin,thereby reducing or preventing unwanted side-effects associated withtoxin dispersal into non-targeted areas.

One parameter that is often used to measure the binding affinity isdefined as binding K_(D). Because K_(D) is defined as the ratio betweendissociation constant (K_(off)) and association constant (K_(on)), thelower the K_(D) value, the higher the binding affinity.

As the term is used herein, “significantly enhanced binding” when usedto describe the binding affinity of a C. botulinum neurotoxin moleculeor BoNT/B-Hc binding fragment thereof, of the present invention to aspecific receptor (e.g., human Syt II or human Syt I), refers to anincrease in binding affinity for a specific receptor that issubstantially increased (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% of the binding affinity of the wild type molecule) ascompared to the non-substituted version of the molecule. In oneembodiment, the enhanced binding of the substituted molecule isdemonstrated by a binding affinity that is an order of magnitude or morethan the binding affinity of the non-substituted molecule (e.g., theneurotoxin with a naturally occurring BoNT H_(C) molecule). Put anotherway, the K_(D) of the substituted molecule is an order of magnitude ormore lower than the K_(D) of the non-substituted molecule In oneembodiment, the enhanced binding is demonstrated by a binding affinitythat is significantly higher (e.g., 1.5×, 2.0×, 2.5×, 3.0×, etc.) thanthe binding affinity of the non-substituted molecule. Put another way,the K_(D) of the substituted molecule is significantly lower (e.g.,1.5×, 2.0×, 2.5×, 3.0×, etc.) than the K_(D) of the non-substitutedmolecule. In one embodiment, the enhanced binding demonstrated is in therange of about 0.59 μM to 5.82 μM K_(D). In one embodiment, the enhancedbinding demonstrated is in the range of about 0.59 μM to 4.3 μM K_(D).In one embodiment, the enhanced binding is demonstrated by a K_(D)≤5.82μM. In one embodiment, the enhanced binding is demonstrated by aK_(D)≤4.30 μM. In one embodiment, the enhanced binding is demonstratedby a K_(D)≤2.9 μM. In one embodiment, the enhanced binding isdemonstrated by a K_(D) of about 0.59 μM.

In one embodiment, the K_(D) of the substituted molecule for human SytII is ≤10 μM, preferably ≤9, 8, 7, 6, 5, 4, 3 or 2 μM, more preferably≤1 or 0.6 μM. In one embodiment, the K_(D) of the substituted moleculefor human Syt I is ≤10 μM, preferably ≤9, 8, 7, 6, 5, 4 μM, morepreferably ≤3 μM. In one embodiment, the K_(D) of the substitutedmolecule for human Syt II is ≤7 μM and the K_(D) of the substitutedmolecule for human Syt I is ≤6 μM. In a preferred embodiment, the K_(D)of the substituted molecule for human Syt II is ≤1 μM and the K_(D) ofthe substituted molecule for human Syt I is ≤3 μM.

In one embodiment, enhanced binding results in detectable binding tohuman Syt I in the absence of co-receptor gangliosides, such asexemplified by the experiments described herein.

The term “significantly enhanced binding” or “significantly reducedbinding” when used to describe the binding affinity of a BoNT/B-H_(C)binding fragment produced by the point mutations described herein refersto an increase or decrease, respectively, in binding affinity of themodified binding domain (e.g., expressed as an isolated fragment or inthe context of the larger polypeptide) to a specific receptor (e.g.,human Syt II or human Syt I) as discussed directly above.

As the term is used herein, “significantly reduced binding” when used todescribe the binding affinity of botulinum neurotoxin molecule orbinding fragment thereof (e.g., BoNT/B-Hc), of the present invention toa specific receptor (e.g., human Syt I or human Syt II), refers to adecrease in binding affinity for the specific receptor that issubstantially decreased (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% of the binding affinity of the wild type molecule) ascompared to the non-substituted version of the molecule. In oneembodiment, the decreased binding of the substituted molecule results ina binding affinity that is an order of magnitude or more less than thebinding affinity of the non-substituted neurotoxin (e.g., the neurotoxinwith a naturally occurring BoNT H_(C) molecule). Put another way, theK_(D) of the substituted molecule is an order of magnitude or morehigher than the K_(D) of the non-substituted neurotoxin. In oneembodiment, the reduced binding of the substituted molecule results in abinding affinity that is significantly lower (e.g., 1.5×, 2.0×, 2.5×,3.0×, etc.) than the binding affinity of the non-substituted molecule.Put another way, the K_(D) of the substituted molecule is significantlyhigher (e.g., 1.5×, 2.0×, 2.5×, 3.0×, etc.) than the K_(D) of thenon-substituted molecule. In one embodiment, significantly reducedbinding results in loss of detectable binding to human Syt I in theabsence of co-receptor gangliosides.

As used herein, the term “botulinum neurotoxin” means any polypeptidethat can execute the overall cellular mechanism whereby a C. botulinumtoxin enters a neuron and inhibits neurotransmitter release. Thismechanism encompasses the binding of a C. botulinum toxin to a low orhigh affinity receptor complex, the internalization of the toxin, thetranslocation of the toxin light chain into the cytoplasm and theenzymatic modification of a C. botulinum toxin substrate. The term“polypeptide” and “protein” are used interchangeably herein, inreference to the C. botulinum neurotoxin molecule and fragments thereof.

A “modified receptor binding domain” or “modified H_(C)”, as the term isused herein, facilitates the binding of the C. botulinum neurotoxinmolecule in which it is comprised, to a receptor for C. botulinumneurotoxin located on the surface of a target cell. Such a molecule istypically generated through genetic recombination technology. Themodified H_(C) has a binding activity for the receptor for C. botulinumneurotoxin located on the surface of a target cell. As used herein, theterm “binding activity” means that one molecule is directly orindirectly contacting another molecule via at least one intermolecularor intramolecular force, including, without limitation, a covalent bond,an ionic bond, a metallic bond, a hydrogen bond, a hydrophobicinteraction, a van der Waals interaction, and the like, or anycombination thereof “Bound” and “bind” are considered terms for binding.

As used herein, the term “C. botulinum toxin protease domain” means a C.botulinum toxin domain that can execute the enzymatic targetmodification step of the intoxication process. Thus, a C. botulinumtoxin protease domain specifically targets a C. botulinum toxinsubstrate and encompasses the proteolytic cleavage of a C. botulinumtoxin substrate, such as, e.g., SNARE proteins like a SNAP-25 substrate,a VAMP substrate and a Syntaxin substrate.

Non-limiting examples of C. botulinum toxin protease domains areprovided in Tables 1 and 2.

As used herein, the term “C. botulinum toxin translocation domain” or“H_(N)” means a C. botulinum toxin domain that can execute thetranslocation step of the intoxication process that mediates C.botulinum toxin light chain translocation. Thus, a H_(N) facilitates themovement of a C. botulinum toxin light chain across a membrane andencompasses the movement of a C. botulinum toxin light chain through themembrane an intracellular vesicle into the cytoplasm of a cell.Non-limiting examples of a H_(N) include a BoNT/A H_(N), a BoNT/B H_(N),a BoNT/C1 H_(N), a BoNT/D H_(N), a BoNT/E H_(N), a BoNT/F H_(N), and aBoNT/G H_(N), the amino acid sequences of which are provided in Table 1and FIGS. 12, 14-20.

As used herein, the term “C. botulinum receptor-binding domain” issynonymous with “H_(C) domain” and means any naturally occurring C.botulinum receptor binding domain that can execute the cell binding stepof the intoxication process, including, e.g., the binding of the C.botulinum toxin to a C. botulinum toxin-specific receptor system locatedon the plasma membrane surface of a target cell. It is envisioned thatreplacement of the binding activity can be achieved by, e.g., replacingthe entire C. botulinum H_(c) domain with a modified (e.g., enhanced)H_(C) domain.

As used herein, the term “C. botulinum toxin target cell” means a cellthat is a naturally occurring cell that a naturally occurring C.botulinum toxin is capable of intoxicating, including, withoutlimitation, motor neurons; sensory neurons; autonomic neurons; such as,e.g., sympathetic neurons and parasympathetic neurons; non-peptidergicneurons, such as, e.g., cholinergic neurons, adrenergic neurons,noradrenergic neurons, serotonergic neurons, GABAergic neurons; andpeptidergic neurons, such as, e.g., Substance P neurons, Calcitonin GeneRelated Peptide neurons, vasoactive intestinal peptide neurons,Neuropeptide Y neurons, cholecystokinin neurons.

By “isolated” is meant a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source orsurroundings, e.g. from flanking DNA or from the natural source of theDNA, or from flanking amino acids.

The term “purified” is used to refer to a substance such as apolypeptide that is “substantially pure”, with respect to othercomponents of a preparation (e.g., other polypeptides). It can refer toa polypeptide that is at least about 50%, 60%, 70%, or 75%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% pure, with respect to other components.Recast, the terms “substantially pure” or “essentially purified”, withregard to a polypeptide, refers to a preparation that contains fewerthan about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, mostpreferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of oneor more other components (e.g., other polypeptides or cellularcomponents).

The term “conservative” or “conservative substitution mutation” as usedherein refers to a mutation where an amino acid is substituted foranother amino acid that has similar properties, such that one skilled inthe art of peptide chemistry would expect the secondary structure,chemical properties, and/or hydropathic nature of the polypeptide to besubstantially unchanged. The following groups of amino acids have beenhistorically substituted for one another as conservative changes: (1)ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, try, thr; (3)val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp,his. Other commonly accepted conservative substitutions are listedbelow:

Conservative Conservative Residue Substitutions Residue SubstitutionsAla Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile AspGlu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu AspTrp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val

The term “substitution mutation” without the reference to a specificamino acid, may include any amino acid other than the wild type residuenormally found at that position. Such substitutions may be replacementwith non-polar (hydrophobic) amino acids, such as glycine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, andproline. Substitutions may be replacement with polar (hydrophilic) aminoacids such as serine, threonine, cysteine, tyrosine, asparagine, andglutamine. Substitutions may be replacement with electrically chargedamino acids e.g., negatively electrically charged amino acids such asaspartic acid and glutamic acid and positively electrically chargedamino acids such as lysine, arginine, and histidine.

The substitution mutations described herein will typically bereplacement with a different naturally occurring amino acid residue, butin some cases non-naturally occurring amino acid residues may also besubstituted. Non-natural amino acids, as the term is used herein, arenon-proteinogenic (i.e., non-protein coding) amino acids that eitheroccur naturally or are chemically synthesized. Examples include but arenot limited to β-amino acids (β3 and β2), homo-amino acids, proline andpyruvic acid derivatives, 3-substituted alanine derivatives, glycinederivatives, ring-substituted phenylalanine and tyrosine derivatives,linear core amino acids, diamino acids, D-amino acids, and N-methylamino acids. In some embodiments, the amino acid can be substituted orunsubstituted. The substituted amino acid or substituent can be ahalogenated aromatic or aliphatic amino acid, a halogenated aliphatic oraromatic modification on the hydrophobic side chain, or an aliphatic oraromatic modification.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically significant reduction in one ormore symptoms of the condition when administered to a typical subjectwho has the condition. A therapeutically significant reduction in asymptom is, e.g. about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 100%, or more ascompared to a control or non-treated subject.

The term “treat” or “treatment” refers to therapeutic treatment whereinthe object is to eliminate or lessen symptoms. Beneficial or desiredclinical results include, but are not limited to, elimination ofsymptoms, alleviation of symptoms, diminishment of extent of condition,stabilized (i.e., not worsening) state of condition, delay or slowing ofprogression of the condition.

As used herein, a “subject” refers to a human or animal. Usually theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female. A subject can be a fully developedsubject (e.g., an adult) or a subject undergoing the developmentalprocess (e.g., a child, infant or fetus).

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders associated with unwanted neuronal activity. In addition, themethods and compositions described herein can be used to treatdomesticated animals and/or pets.

Embodiments

The observation that BoNT/B is less specific and potent in humans due toits inability to bind human Syt II, may explain why comparatively higherdoses are required than BoNT/A. Higher BoNT/B doses correspond toincreased chances for triggering antibody responses and for seriousside-effects to occur. Therefore, improved binding of BoNT/B to thehuman receptor Syt II, to increase its efficacy and specificity totarget human neurons should allow a reduced amount of the toxin dosesused in therapeutic applications.

Aspects of the invention arise from the finding that modifying theprotein sequence of BoNT/B-H_(C) modifies binding of the fragmentcontaining the receptor binding domain, to the human Syt II receptor.Specific modifications have been identified that enhance binding,thereby generating a domain that binds human Syt II with high-affinity.The modified BoNT/B-H_(C), when in the context of a full length BoNTprotein, retains these binding properties. Incorporation of a modifiedreceptor binding domain with enhanced binding, into a moleculecomprising the other BoNT domains, thereby generates a full length BoNTmolecule with similarly enhanced receptor binding. As such, new versionsof BoNT with high-affinity binding to human Syt II are generated. BoNTwith significantly enhanced binding can be used in similar therapies,albeit at lower doses than presently available BoNT molecules, thusproviding safer methods of treatment.

The BoNT polypeptides, including full-length BoNT polypeptides and BoNTpolypeptide fragments or domains described herein (e.g., the modifiedBoNT/H_(C)), and nucleic acid molecules which encode them, areexplicitly encompassed in the invention. These polypeptides and nucleicacid molecules can be generated by recombinant DNA procedures known inthe art. Such polypeptides are typically referred to as “recombinantpolypeptides” or “recombinant nucleic acids”.

Botulinum Neurotoxin Protein

One aspect of the invention relates to a botulinum neurotoxin (BoNT)protein comprising a modified receptor binding domain (e.g., ofClostridial botulinum serotype B), as described herein. The BoNT proteinfurther comprises a protease domain, a translocation domain, and aprotease cleavage site. Typically these are arranged in a linearamino-to-carboxyl single polypeptide order of the protease domain, theprotease cleavage site, the translocation domain and the modifiedreceptor binding domain. However, different arrangements of the variousdomains are expected to function adequately. In one embodiment, themodified receptor binding domain comprises one or more substitutionmutations which lead to significantly enhanced binding to the human SytI receptor and/or the human Syt II receptor.

The BoNT protein may further comprise a domain that is useful forpurification of the molecule such as a hexahistidine tag (His6) (SEQ IDNO: 13), or an epitope tag such as a hemaglutinin (HA) tag (YPYDVPDYA(SEQ ID NO: 12)). Various such domains are known and used in the art forsuch purposes.

BoNT has the overall structure shown in FIG. 1B. BoNT is comprised ofthree domains, each domain having a specific and independent function: aprotease domain (also referred to as the light chain), a translocationdomain (H_(N)), and a receptor-binding domain (H_(C)). Domains of thevarious strains of C. botulinum neurotoxin have been shown to be largelyinterchangeable (as demonstrated by naturally occurred chimeric toxinssuch as BoNT/CD, which is composed of the light chain and H_(N) ofBoNT/C, with the H_(C) of BoNT/D³⁴, in U.S. Pat. No. 8,052,979,incorporated herein by reference). The protein can be in single chainform or di-chain form. The di-chain form results from the naturallyoccurring protease processing of a protease cleavage site locatedbetween the protease domain and the translocation domain. The protein ismaintained in the di-chain form following protease processing by thepresence of a di-sulfide bond.

Strains of Clostridia botulinum produce seven antigenically-distincttypes of botulinum toxins, which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and/F), animals (BoNT/C1and/D), or isolated from soil (BoNT/G). While all seven BoNT serotypeshave similar structure and pharmacological properties, each alsodisplays heterogeneous bacteriological characteristics. The geneticdiversity of the C. botulinum strains is described in detail in Hill etal. (Journal of Bacteriology, Vol. 189, No. 3, p. 818-832 (2007))³⁵, thecontents of which are incorporated herein by reference. In oneembodiment, the BoNT of the invention has domains which are all of thesame serotype (A, B, C, D, E, F or G). In one embodiment, one or more ofthose domains of the same serotype differ as to their strain and/orsubtype.

Toxins from the various C. botulinum serotypes/strains share the samefunctional domain organization and overall structural architecture. C.botulinum toxins are each translated as a single chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease, suchas, e.g., an endogenous C. botulinum toxin protease or anaturally-occurring proteases produced in the environment. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by a single disulfide bond and noncovalentinteractions. Each mature di-chain molecule comprises three functionallydistinct domains: 1) a proteolytic domain located in the LC thatincludes a metalloprotease region containing a zinc-dependentendopeptidase activity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC (H_(N)) that facilitatesrelease of the LC from intracellular vesicles into the cytoplasm of thetarget cell; and 3) a binding domain found within the carboxyl-terminalhalf of the HC that determines the binding activity and bindingspecificity of the toxin to the receptor complex located at the surfaceof the target cell. The locations of the specific domains within thetoxin of various serotypes/strains are provided in Table 1:

TABLE 1 C. botulinum toxin domains from various serotypes/strains ToxinLC H_(N) H_(C) BoNT/A M1-K448 A449-K871 N872-L1296 BoNT/B M1-K441A442-S858 E859-E1291 BoNT/C1 M1-K449 T450-N866 N867-E1291 BoNT/D M1-R445D446-N862 S863-E1276 BoNT/E M1-R422 K423-K845 R846-K1252 BoNT/F M1-K439A440-K864 K865-E1274 BoNT/G M1-K446 S447-S863 N864-E1297

Complete amino acid sequences of the toxins are provided in FIGS. 14-21.

The binding, translocation and protease activity of these threefunctional domains are all necessary for toxicity. The overall cellularintoxication mechanism whereby C. botulinum toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Without wishing to be bound by theory, the intoxicationmechanism involves at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) protease targetmodification. The process is initiated when the H_(C) domain of a C.botulinum toxin binds to a toxin-specific receptor located on the plasmamembrane surface of a target cell. The binding specificity of a receptorcomplex is thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation step istriggered by the acidification of the vesicle compartment. Oncetranslocated, light chain endopeptidase of the toxin is released fromthe intracellular vesicle into the cytosol where it specifically targetsone of three proteins known as the core components of theneurotransmitter release apparatus (vesicle-associated membrane protein(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa(SNAP-25) and Syntaxin). These core components are necessary forsynaptic vesicle docking and fusion at the nerve terminal and constitutemembers of the soluble N-ethylmaleimide-sensitive factor-attachmentprotein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in thecarboxyl-terminal region, releasing a nine or twenty-six amino acidsegment, respectively, and BoNT/C1 also cleaves SNAP-25 near thecarboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F andBoNT/G, and tetanus toxin, act on the conserved central portion of VAMP,and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1cleaves syntaxin at a single site near the cytosolic plasma membranesurface. The selective proteolysis of synaptic SNAREs accounts for theblock of neurotransmitter release caused by C. botulinum toxins in vivo.The SNARE protein targets of C. botulinum toxins are common toexocytosis in a variety of non-neuronal types; in these cells, as inneurons, light chain peptidase activity inhibits exocytosis, see, e.g.,Yann Humeau et al., How Botulinum and Tetanus Neurotoxins BlockNeurotransmitter Release, 82(5) Biochimie. 427-446 (2000); KathrynTurton et al., Botulinum and Tetanus Neurotoxins: Structure, Functionand Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxinsin Neurons, 11(9) Trends Microbiol. 431-437, (2003).

Domains and Chimeric Neurotoxins

The botulinum neurotoxin of the present invention comprises a modifiedreceptor binding domain (H_(C)). The modified receptor binding domainexhibits significantly enhanced binding to one or more human receptorstypically bound and utilized by one or more C. botulinum toxin strains(e.g., SytII, SytI). The modified receptor binding domain can be of anyserotype (A, B, C, D, E, F or G), strain or subtype (as describedherein). This may be the same or different serotype, strain/subtype asone or more other domains within the BoNT. Examples of specific modifiedreceptor binding domains are provided herein. The isolated modifiedreceptor binding domain polypeptide described herein is also encompassedby the present invention, as is the isolated nucleic acid molecule bywhich it is encoded. In one embodiment, the H_(C) is serotype B. In oneembodiment, the H_(C) is serotype A.

The botulinum neurotoxin of the present invention also comprises aprotease domain, also referred to in the art as a light chain variant.The light chain variant may be a naturally occurring light chainvariant, such as, e.g., C. botulinum toxin light chain isoforms and C.botulinum toxin light chain subtypes; or a non-naturally occurring C.botulinum toxin light chain variant, such as, e.g., conservativesubstitution C. botulinum toxin light chain variants. The proteasedomain can be of any serotype (A, B, C, D, E, F or G), strain or subtype(as described herein). This may be the same or different serotype,strain/subtype as one or more other domains within the BoNT. In oneembodiment, the protease domain is serotype B. In one embodiment, theprotease domain is serotype A.

The botulinum neurotoxin of the present invention also comprises a toxintranslocation domain (H_(N)). The toxin translocation domain can be ofany serotype (A, B, C, D, E, F or G), strain or subtype (as describedherein). This may be the same or different serotype, strain/subtype asone or more other domains within the BoNT. In one embodiment, the H_(N)is serotype B. In one embodiment, the H_(N) is serotype A.

The various domains described herein (e.g., H_(N), H_(C), or proteasedomain) include, without limitation, naturally occurring variants, suchas, e.g., isoforms and subtypes; non-naturally occurring variants, suchas, e.g., conservative substitution mutations. Non-naturally-occurringvariants, refers to a domain that has at least one amino acid changefrom the corresponding region of the reference sequences (e.g., fromTable 1 or FIGS. 14, 16-23) and can be described in percent identity tothe corresponding region of that reference sequence.

It is recognized by those of skill in the art that within each serotypeof C. botulinum toxin there can be naturally occurring C. botulinumdomain variants that differ somewhat in their amino acid sequence, andalso in the nucleic acids encoding these proteins. A naturally occurringC. botulinum toxin domain (e.g., light chain, H_(N) or H_(C)) variantenvisioned for use in the generation of the BoNT of the presentinvention can function in substantially the same manner as the referenceC. botulinum toxin domain on which the naturally occurring C. botulinumdomain variant is based, and can be substituted for the reference C.botulinum toxin domain in any aspect of the present invention.

A non-limiting example of a naturally occurring C. botulinum toxindomain variant is a C. botulinum toxin domain isoform such as, e.g., aBoNT/A domain isoform, a BoNT/B domain isoform, a BoNT/C1 domainisoform, a BoNT/D domain isoform, a BoNT/E domain isoform, a BoNT/Fdomain isoform, and a BoNT/G domain isoform. A C. botulinum toxin domainisoform can function in substantially the same manner as the referenceC. botulinum toxin domain on which the C. botulinum toxin domain isoformis based, and can be substituted for the reference C. botulinum toxindomain in any aspect of the present invention.

Another non-limiting example of a naturally occurring C. botulinum toxindomain variant is a C. botulinum toxin domain subtype such as, e.g., adomain from subtype BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5; adomain from subtype BoNT/B1, BoNT/B2, BoNT/B3, BoNT/B4, BoNT/B5,BoNT/B6, BoNT/B7; a domain from subtype BoNT/C1-1, BoNT/C1-2, BoNT/D-C;a domain from subtype BoNT/E1, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5,BoNT/E6, BoNT/E7, BoNT/E8; and a domain from subtype BoNT/F1, BoNT/F2,BoNT/F3, BoNT/F4, BoNT/F5, BoNT/F6, BoNT/F7. A C. botulinum toxin domainsubtype can function in substantially the same manner as the referenceC. botulinum toxin domain on which the C. botulinum toxin domain subtypeis based, and can be substituted for the reference C. botulinum toxindomain in any aspect of the present invention.

As used herein, the term “non-naturally occurring variant” (e.g., C.botulinum toxin light chain variant, H_(C) and H_(N)) means a C.botulinum domain produced with the aid of human manipulation, including,without limitation, domains produced by genetic engineering using randommutagenesis or rational design and C. botulinum domains produced bychemical synthesis. Non-limiting examples of non-naturally occurring C.botulinum domain variants include, e.g., conservative C. botulinumdomain variants. As used herein, the term “conservative C. botulinumdomain variant” means a C. botulinum domain that has at least one aminoacid substituted by another amino acid or an amino acid analog that hasat least one property similar to that of the original amino acid fromthe reference C. botulinum domain sequence (e.g., Table 1 and FIGS. 12,14-21). The variant may have one, two, three, four, five or moreconservative amino acid substitutions compared to the reference domainsequence. Examples of properties include, without limitation, similarsize, topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservative C.botulinum domain variant can function in substantially the same manneras the reference C. botulinum toxin domain on which the conservative C.botulinum toxin domain variant is based, and can be substituted for thereference C. botulinum domain in any aspect of the present invention.

A non-naturally occurring C. botulinum toxin domain variant maysubstitute one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, ormore) from the reference C. botulinum toxin domain on which thenaturally occurring C. botulinum toxin domain is based. A non-naturallyoccurring C. botulinum toxin domain variant can also possess 95% or more(e.g., 96%, 97%, 98% or 99%) amino acid identity to the reference C.botulinum toxin domain on which the naturally occurring C. botulinumdomain variant is based.

Various non-naturally occurring C. botulinum neurotoxins or specificdomains thereof, are described in International Patent PublicationsWO95/32738, WO96/33273, WO98/07864 and WO99/17806, each of which isincorporated herein by reference. The C. botulinum neurotoxin orspecific domain thereof described herein will typically containnaturally occurring amino acid residues, but in some cases non-naturallyoccurring amino acid residues may also be present. Therefore, so-called“peptide mimetics” and “peptide analogues”, which may include non-aminoacid chemical structures that mimic the structure of a particular aminoacid or peptide, may also be used within the context of the invention.Such mimetics or analogues are characterized generally as exhibitingsimilar physical characteristics such as size, charge or hydrophobicity,and the appropriate spatial orientation that is found in their naturalpeptide counterparts. A specific example of a peptide mimetic compoundis a compound in which the amide bond between one or more of the aminoacids is replaced by, for example, a carbon-carbon bond or othernon-amide bond, as is well known in the art (see, for example Sawyer, inPeptide Based Drug Design, pp. 378-422, ACS, Washington D.C. 1995).

In one aspect of the invention, the botulinum neurotoxin (BoNT) of thepresent invention comprises a modified receptor binding domain of C.botulinum serotype B (BoNT/B-H_(C)). The modified BoNT/B-H_(C) comprisesone or more substitution mutations which lead to significantly enhancedbinding to the human Syt I receptor and/or the human Syt II receptor. Inone embodiment, the BoNT/B-H_(c) is from BoNT/B1 (GenBank access No.:AB232927.1). The amino acid sequence of BoNT/B1-H_(C) Okra strain, usedas the reference template in the present invention is shown in FIG. 12.The generation of B-H_(c) from other strains and subtypes bysubstitution of the amino acids that correspond to the specifiedposition(s) in B1 described herein is also envisioned, as is thegeneration of longer molecules that incorporate the B-H_(c) such ascomplete BoNT or a polypeptide comprising the modified B-H_(c). Suchmolecules are also encompassed in the invention. In one embodiment, theBoNT or the polypeptide of the present invention comprises a modifiedreceptor binding domain which is not a BoNT/B4 strain known as theEklund strain (NCBI Reference Sequence: YP_001893661.1, Genbank Ref:EF051570.1), shown in FIG. 21. In one embodiment, the modified B-Hc isnot of strain 3, 7 or 8.

Toxin diffusion and generation of neutralization antibodies is a problemassociated with but not limited to BoNT/B, as they are also observedwith BoNT/A. Improvement of the binding affinity of BoNT/A to itsreceptor SV2 would relieve these problems. Because BoNT/B binding to SytI/II has much higher affinity than BoNT/A binding to SV2^(14,20,26,27),a modified BoNT/B receptor binding domain (BoNT/B-H_(C)) with theability to bind human Syt II can also be used to replace BoNT/A-H_(C) togenerate a modified chimeric BoNT/A which may have greater efficacy andspecificity for human neurons than WT BoNT/A.²⁸ ²⁹ ³⁰

It is further envisioned that the modified H_(C) described above (e.g.,BoNT/B-H_(C)) can be utilized to replace the H_(C) of all other BoNTserotypes/strains. As such, another aspect of the invention is a BoNTpolypeptide comprising a modified receptor biding domain (H_(C)) ofserotype (A, B, C, D, E, F or G), joined with one or more domains of adifferent serotype (A, B, C, D, E, F or G), strain or subtype to therebyproduce a chimeric neurotoxin. The H_(C) region of each BoNTs is welldefined and respective H_(C) regions can be exchanged in the BoNTmolecule (e.g., via genetic engineering). Such manipulation is routinelyperformed by the skilled practitioner via standard PCR fusion of DNAencoding BoNT/B-H_(C) with the light chain (LC) and translocation domain(H_(N)), or H_(N)-LC, of other BoNTs, which has been well-established inthe art. In addition, the replacement may also be performed using theC-terminal part of BoNT/B-H_(C) (designated as H_(CC)), which is theregion containing the binding site for protein receptors andgangliosides in each BoNT. The resulting chimeric toxins will have theability to target human neurons via binding to human Syt I/II. As anon-limiting example, modified BoNT/B-H_(C) (or BoNT/B-H_(CC)) can beused to replace the H_(C) (or H_(CC)) of BoNT/A. The resultingpolypeptides are encompassed by the instant invention. These chimerictoxins may have a higher efficacy and specificity targeting humanneurons than WT BoNT/A. Such a chimeric BoNT/A toxin can be used fortherapeutic applications in humans and offers significant improvementsover WT BoNT/A.

One aspect of the invention relates to an isolated, purified modifiedreceptor binding domain polypeptide as described herein. In oneembodiment, the modified receptor binding domain is BoNT/B-H_(C) (e.g.,from BoNT/B1). In one embodiment, the B-H_(c) is generated from adifferent strain and/or subtype of BoNT by substitution of the aminoacids that correspond to the specified position(s) in B1 describedherein. In one embodiment, the H_(C) is subtype B2, B3, B4, B5, B6 orB7. In one embodiment, the modified receptor binding domain is not fromBoNT/B4 (the Eklund strain). In one embodiment, the modified B-Hc is notof strain 3, 7 or 8.

The present invention encompasses mutant full-length BoNT that containsa modified H_(C) (e.g., B-H_(C)) with amino acid substitutions asdescribed herein, for therapeutic applications in humans. In oneembodiment, the full-length BoNT contains a modified H_(C) (e.g.,B-H_(C)) with an amino acid substitution at one or combinations of theamino acid residues corresponding to position E1191, S1199, Y1183, andW1178 of B1 (e.g., selected from those listed in Table 2). In oneembodiment, the BoNT contains a modified H_(C) (e.g., B-H_(C)) with asingle amino acid substitution as described herein. In one embodiment,the BoNT contains a modified H_(C) (e.g., B-H_(C)) with two amino acidsubstitutions as described herein. In one embodiment, the BoNT containsa modified H_(C) (e.g., B-H_(C)) with three amino acid substitutions asdescribed herein. The mutations can be made in the same manner asdisclosed herein for BoNT/H_(C), (e.g., using any one of BoNT/B subtypesor any serotype as template). In one embodiment, the mutant full-lengthBoNT contains a modified Hc that is not from BoNT/B4 (the Eklundstrain). In one embodiment, the modified B-Hc is not of strain 3, 7 or8.

The resulting BoNT toxin can have significantly enhanced binding tohuman Syt II, and therefore will achieve higher efficacy and specificityto target human neurons than WT BoNT. The resulting mutant may furthermaintain similar binding to human SytI, have significantly enhancedbinding to human SytI, or have significantly reduced binding to humanSytI.

Polypeptides and Chimeric Polypeptides

Another aspect of the invention relates to a polypeptide comprising themodified receptor binding domain (e.g., of serotype A, B, C, D, E, F orG). In one embodiment, the modified Hc, in the context of thepolypeptide, has significantly enhanced binding to human Syt II and/orSyt I, as compared to the analogous polypeptide with WT amino acids atthe specific positions modified in the Hc (the wild type counterpart).In one embodiment, the modified receptor binding domain is BoNT/B-H_(C)(e.g., from BoNT/B1). In one embodiment, the modified H_(c) is generatedfrom a different serotype, strain and/or subtype of BoNT by substitutionof the amino acids that correspond to the specified position(s) in B1described herein. In one embodiment, the modified Hc is not from BoNT/B4(the Eklund strain). In one embodiment, the modified B-Hc is not ofstrain 3, 7 or 8. In one embodiment, the modified receptor bindingdomain has one or more modifications (amino acid substitutions) in theC-terminal part of BoNT/B-H_(C) (designated as H_(CC)), which is theregion containing the binding site for protein receptors andgangliosides in each BoNT. In one embodiment, the resulting polypeptidehas the ability to target human neurons via binding to human SytI/II.

The polypeptide comprising the modified receptor binding domain may be afusion protein of the receptor binding domain and another functionalpolypeptide (e.g., a functional polypeptide utilized in a 2-hybridsystem (bait or prey such as T18 or T25, or glutathione S transferase(GST)). This can also be referred to as a chimeric polypeptide molecule.Alternatively, it may be a fusion of the modified receptor bindingdomain and a polypeptide tag for identification purposes and/orpurification purposes (e.g., a hexahistidine tag (His6) (SEQ ID NO: 13),or an epitope tag such as a hemaglutinin (HA) tag (YPYDVPDYA (SEQ ID NO:12)), or GST), many of which are known and commonly used in the art. Thefusion may have a stretch of amino acids between the respectivefunctional domains that facilitates linkage, serves as a cleavage site,or to preserve independent conformation and/or function. In oneembodiment, the linkage preserves the function of the modified receptorbinding domain. In one embodiment, the linkage masks the function of themodified receptor binding domain. Another aspect of the inventionrelates to a nucleic acid molecule which encodes any one of suchpolypeptides.

The modified BoNT/H_(C) (e.g., B-H_(C)) can be linked to other agents(e.g., proteins, small molecules, short polypeptides, nucleic acids)covalently (e.g., as a fusion protein) or non-covalently. As such,another aspect of the invention relates to a chimeric moleculecomprising a first portion that is a modified receptor binding domain ofC. botulinum (e.g., serotype B), as described herein, linked to a secondportion. The second portion of the molecule can be a bioactive (or“biologically active”) molecule such as a therapeutic agent (e.g., apolypeptide or non-polypeptide drug such as a small molecule or nucleicacid). Linkage of the first and second portions of the molecule can becovalent (e.g., in the form of a fusion protein) or non-covalent.Methods of such linkage are known in the art and can readily be appliedby the skilled practitioner. One such use of a chimeric molecule of theinvention is as a delivery tool to target neurons in humans. Forexample, the modified BoNT/H_(C) can be linked to other therapeuticagents, to serve as the targeting vehicle to deliver the therapeuticagents to neurons in humans by binding to human Syt I and/or Syt II.

Modifications of the Receptor Binding Domain (Hc)

As discussed herein, the invention relates to a modified Hc andpolypeptides comprising the modified Hc (e.g., BoNT or a fusion orchimeric polypeptide). The modification of the H_(C) amino acid sequenceused to generate these various polypeptides of the invention can beperformed by various methods known to the skilled practitioner. Examplesinclude, without limitation, targeted mutagenesis (site-directedmutagenesis) or random mutagenesis of each amino acid residue within theregion known for binding Syt I/II. These Syt binding regions are welldefined by previous studies relating to mouse or rat Sytreceptors^(1,29,36,31,32) but have not been clearly determined forinteractions between BoNT/B-H_(c) and human Syt receptors. Differentsubtypes of BoNT/B, or other serotypes which bind to Syt I/II (D-C orG), can be used as the template to create the same or similar mutationsby generating corresponding mutations described herein for B1-H_(C). Thecorresponding position for selected residues to be mutated can bereadily identified by sequence alignment with the B1 subtype. Theresulting polypeptide products are encompasses by the instant invention,as are polypeptides comprising said products and nucleic acid moleculesencoding said polypeptides and products.

Amino acid sequence modifications of H_(C) to produce the modifiedreceptor binding domain can be mutation of a single residue to adifferent amino acid (single site substitution), mutation of multipleresidues at the same time (multiple sites substitution), deletion of oneor more residues (deletion), and insertion of one or more residues(insertion), as well as combinations thereof. Methods for mutatingproteins are well-known in the art (e.g., targeted single site andmultiple sites substitutions on the DNA encoding the BoNT/H_(C)sequence).

In one embodiment, one or more residues in H_(c) that either contactrodent Syt II or the surrounding regions, based on previous literatureson BoNT/B receptor binding domain²⁹ and reported BoNT/B-Syt II structure(PDB ID: 2NM1)^(31,32) are modified. These include, without limitationthose positions that correspond to position Y1181, P1197, A1196, F1204,F1194, P1117, W1178, Y1183, V1118, S1116, K1113, K1192, S1199, S1201,E1191, E1245, Y1256, D1115, E1203 of BoNT/B-B1. In one embodiment, oneor more of these residues is systemically replaced with other aminoacids. Combinations of various modifications are also envisioned,including, without limitation, mutations of two or more recitedpositions, to any variety of amino acids, such as those recited herein.

Multiple site substitutions (e.g., 2 or 3) can be generated by combiningmutations in these identified key residues. Such multiple sitesubstitution mutants may have even further enhanced binding to h-Syt IIand may further maintain or have enhanced binding to human Syt I. In oneembodiment, the modified H_(C) has a first amino acid substitution thatcorresponds to E1191 of serotype B, strain 1, substituted with either Q,M, C, V, L, or Y. In one embodiment, the modified H_(C) has a secondsite substitution that corresponds to either S1199W, S1199E, S1199H,S1199Y, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, S1199F or S1199Lof serotype B, strain 1. In one embodiment, the modified H_(C) has amultiple site substitution that corresponds to either E1191M and S1199W,E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C andW1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, orE1191V and W1178Q, of serotype B, strain 1.

In one embodiment, the modified receptor binding domain comprises asubstitution mutation at a position corresponding to Y1181, P1197,A1196, F1204, F1194, P1117, W1178, Y1183, V1118, S1116, K1113, K1192,S1199, S1201, E1191, E1245, D1115, E1203 or Y1256 of serotype B,strain 1. In one embodiment, the substitution mutation is A, R, N, D, C,Q, E, G, H, I, L, K, M, F, P, T, W, Y or V substituted for S. In oneembodiment, the position corresponds to S1199 or S1201 of serotype B,strain 1. In one embodiment, the substitution mutation is W, E or H. Inone embodiment, the substitution mutation is at S1199, and is W, E or H.In one embodiment, the substitution mutation is at S1201 and is V. Inone embodiment, the positions corresponds to W1178, Y1183, or E1191. Inone embodiment, the position corresponds to W1178 and the substitutionmutation is Y, Q, A or S. In one embodiment, the position correspond toY1183 and the substitution mutation is C or P. In one embodiment, theposition corresponds to E1191 and the substitution mutation is C, V, L,or Y.

A modified H_(C) with a single amino acid substitution mutation shownbelow in Table 2 is also environed, as is its incorporation in thevarious polypeptides described herein.

TABLE 2 E1191M Y1183C S1199E E1191Q Y1183P S1199H E1191C Y1183M S1199YP1117S A1196Y E1191V S1199W P1117M E1191L W1178Q S1199F P1117Y Y1181ME1191Y W1178Y S1199L E1191T W1178A V1118M E1191I W1178S S1201V

In one embodiment, the H_(C) comprises one or more mutations thatcorrespond to E1191C/V/L/Y (E1191C, E1191V, E1191L, or E1191Y),S1199W/E/H (S1199W, S1199E, or S1199H), W1178Y/Q/A/S (W1178Y, W1178Q,W1178A, or W1178S), and/or Y1183C/P (Y1183C or Y1183P) of serotype B,strain 1 (FIG. 3A, B).

In one embodiment, the H_(C) has two substitution mutations wherein oneis a substitution mutation corresponding to position 1191 selected fromTable 2. In one embodiment, the Hc has enhanced binding to h-Syt IIcompared to the analogous polypeptide having WT amino acids at thespecified positions (also referred to herein as the wild typecounterpart). In one embodiment, the generated mutant also maintains orhas significantly enhanced binding to human Syt I as compared to thewild type counterpart. In particular, the mutation is E1191C, E1191V,E1191L, or E1191Y. More particularly, the mutation that corresponds toposition E1191C, E1191V, E1191L, or E1191Y is combined with a secondmutation (e.g., such as those described herein) to generate a modifiedH_(C) that has enhanced binding to h-Syt II compared to the analogouspolypeptide having WT amino acids at the specified positions (alsoreferred to herein as the wild type counterpart). In one embodiment, thegenerated mutant also maintains or has significantly enhanced binding tohuman Syt I as compared to the wild type counterpart (FIG. 4A). In oneembodiment, the second mutation is a substitution mutation correspondingto a substitution shown for position 1183, 1199, 1201, 1178, 1117, or1181 in Table 2. In one embodiment, the second mutation is S1199W,S1199E, S1199H, S1199Y, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P,S1199F or S1199L. In one embodiment the H_(C) has two substitutionmutations that correspond to those shown for serotype B, strain 1, inTable 3:

TABLE 3 E1191M and S1199Y E1191Q and S1199Y E1191C and S1199Y E1191M andS1199W E1191Q and S1199W E1191C and S1199W E1191M and S1199L E1191Q andS1199L E1191V and S1199Y E1191M and S1199Y E1191Q and S1199Y E1191V andS1199W E1191M and S1199F E1191Q and S1199F E1191M and W1178Q E1191Q andW1178Q E1191C and W1178Q E1191V and W1178Q

In one embodiment, the H_(C) has three substitution mutations, alsoreferred to herein as a triple mutation. In one embodiment of the triplemutation, the first mutation corresponds to E1191Q, E1191M, E1191C,E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, S1199Y, S1199F, S1199L,Y1183C, Y1183P, W1178Y, W1178Q, W1178A or W1178S of serotype B,strain 1. In one embodiment, the second mutation corresponds to E1191Q,E1191M, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, S1199Y,S1199F, S1199L, Y1183C, Y1183P, W1178Y, W1178Q, W1178A or W1178S ofserotype B, strain 1 (exclusive of the position serving as the firstmutation). In one embodiment, the third substitution corresponds toE1191Q, E1191M, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H,S1199Y, S1199F, S1199L, Y1183C, Y1183P, W1178Y, W1178Q, W1178A or W1178Sof serotype B, strain 1 (exclusive of the positions serving as the firstand second mutations). In one embodiment of the invention, the double ortriple mutation does not contain a Q substituted at both positionscorresponding to E1191 and W1178.

In one embodiment of the triple mutation, one mutation is a substitutionmutation that corresponds to a substitution shown for E1191 in Table 2(e.g., E1191Q/M/C/V/L or Y). The second mutation may be a substitutionmutation corresponding to a substitution shown for S1199 in Table 2(e.g., S1199W/E/H/Y/F or L). The third mutation may be a substitutionmutation corresponding to a substitution shown for W1178 in Table 2(e.g., W1178Y/Q/A or S). Alternatively, the third mutation may be asubstitution mutation corresponding to a substitution shown for Y1183 inTable 2. (e.g., Y1183C, Y1183P). In one embodiment, the triple mutationcorresponds to E1191M/S1199W/W1178Q.

Nucleic Acid Molecules

Another aspect of the invention relates to an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes the polypeptidesdescribed herein (e.g., modified receptor binding domain, or apolypeptide comprising the modified receptor binding domain, or thebotulinum neurotoxin comprising the modified receptor binding domain,described herein). In one embodiment, the nucleic acid moleculecomprises the nucleic acid sequence shown in FIG. 13. Such nucleic acidmolecules can be produced by the skilled practitioner, for example byrecombinant DNA techniques. The desired amino acid substitution mutationis made, for example, by modification of the encoding DNA. For example,nucleic acid sequences coding for the polypeptides described herein aregenerated by mutating the nucleic acid codon encoding the specifiedamino acid to the desired amino acid using the genetic code shown below:

TTT Phe TCT Ser TAT Tyr TGT Cys TTC Phe TCC Ser TAC Tyr TGC Cys TTA LeuTCA Ser TGA TTG Leu TCG Ser TGG Trp CTT Leu CCT Pro CAT His CGT Arg CTCLeu CCC Pro CAC His CGC Arg CTA Leu CCA Pro CAA Gln CGA Arg CTG Leu CCGPro CAG Gln CGG Arg ATT Ile ACT Thr AAT Asn AGT Ser ATC Ile ACC Thr AACAsn AGC Ser ATA Ile ACA Thr AAA Lys AGA Arg ATG Met* ACG Thr AAG Lys AGGArg GTT Val GCT Ala GAT Asp GGT Gly GTC Val GCC Ala GAC Asp GGC Gly GTAVal GCA Ala GAA Glu GGA Gly GTG Val GCG Ala GAG Glu GGG Gly

In one embodiment, the nucleic acid sequence is optimized for expressionin E. coli (e.g., the nucleic acid sequence based on GenBank AB232927.1,the relevant portion of which is shown in FIG. 13).

Another aspect of the invention relates to a nucleic acid vectorcomprising the nucleic acid molecule described herein. In one embodimentthe vector is an expression vector. Such an expression vector isreferred to herein as an expression construct, and comprises a nucleicacid molecule disclosed herein operably-linked to the expression vectoruseful for expressing the nucleic acid molecule in a cell or cell-freeextract. A wide variety of expression vectors can be employed forexpressing a nucleic acid molecule encoding a C. botulinum neurotoxin ofthe present invention including, without limitation, a viral expressionvector; a prokaryotic expression vector; eukaryotic expression vectors,such as, e.g., a yeast expression vector, an insect expression vectorand a mammalian expression vector; and a cell-free extract expressionvector. It is further understood that expression vectors useful topractice aspects of these methods may include those which express the C.botulinum neurotoxin under control of a constitutive, tissue-specific,cell-specific or inducible promoter element, enhancer element or both.Non-limiting examples of expression vectors, along with well-establishedreagents and conditions for making and using an expression constructfrom such expression vectors are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN,Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection,making and use of an appropriate expression vector are routineprocedures well within the scope of one skilled in the art and from theteachings herein.

Cells

Another aspect of the invention relates to a cell comprising the nucleicacid molecule or expression construct described herein. The cell can befor propagation of the nucleic acid or for expression of the nucleicacid, or both. Such cells include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacterial cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection, European Collection of Cell Cultures and the GermanCollection of Microorganisms and Cell Cultures. Non-limiting examples ofspecific protocols for selecting, making and using an appropriate cellline are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer AcademicPublishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUESOF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUECULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley andSons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4.sup.th ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3.sup.rd ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra,(2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, OxfordPress, 2.sup.nd ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,supra, (2004). These protocols are routine procedures within the scopeof one skilled in the art and from the teaching herein.

The cell can be for expression of the nucleic acid to thereby generatethe encoded polypeptide. As such, another aspect of the invention is amethod for producing a botulinum neurotoxin protein, an isolated HC, ora polypeptide comprising a modified HC, described herein. Suchpolypeptides are produced by culturing the host cell that has within ita nucleic acid that encodes the polypeptide, in the context of anexpression construct. Culturing is performed under condition suitablefor production of the BoNT polypeptide. The expressed polypeptide can berecovered from the culture, purified and formulated by methods known inthe art. The expressed polypeptide can also be activated as necessary bymethods known in the art. One method of activating the expressedpolypeptide is through cleavage (or nicking) by proteases into an activedichain form. Such methods can be adapted from those known in the art,for example as disclosed by Peter F. Bonventre and Lloyd L. KLempe (J.Bacteriol 1960, 79(1): 23 and Michaeal L. Dekleva and Bibhuti R.DasGupta (Biochemical and Biophysical Research Communications 1989, 162:767-772).

Pharmaceutical Compositions

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising the C. botulinum neurotoxin, or chimeric moleculedescribed herein. In one embodiment, the polypeptide described herein isan active ingredient in a composition comprising a pharmaceuticallyacceptable carrier (referred to herein as a pharmaceutical composition).A “pharmaceutically acceptable carrier” means any pharmaceuticallyacceptable means to mix and/or deliver the targeted delivery compositionto a subject. The term “pharmaceutically acceptable carrier” as usedherein means a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting thesubject agents from one organ, or portion of the body, to another organ,or portion of the body. Each carrier must be “acceptable” in the senseof being compatible with the other ingredients of the composition and iscompatible with administration to a subject, for example a human. Suchcompositions can be specifically formulated for administration via oneor more of a number of routes, such as the routes of administrationdescribed herein. Supplementary active ingredients also can beincorporated into the compositions. When an agent, formulation orpharmaceutical composition described herein, is administered to asubject, preferably, a therapeutically effective amount is administered.As used herein, the term “therapeutically effective amount” refers to anamount that results in an improvement or remediation of the condition.In one embodiment, the pharmaceutical composition is formulated foradministration by injection. In one embodiment, the pharmaceuticalcomposition involves the botulinum neurotoxin encapsulated inmicrospheres. In one embodiment, the pharmaceutical composition involvesthe botulinum neurotoxin formulated for transepithelial delivery. In oneembodiment, the pharmaceutical composition involves the botulinumneurotoxin formulated for slow release.

In one embodiment, the botulinum neurotoxin, polypeptide, or chimericmolecule of the present invention is in the form of a controlled releaseformula. Such compositions and methods for administration are providesin U.S. Patent publication No. 2007/0020295, the contents of which areherein incorporated by reference.

Botulinum neurotoxin can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. The proteolytic strainsthat produce, for example, the botulinum toxin type B serotype may onlycleave a portion of the toxin produced. The exact proportion of nickedto unnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of apreparation of, for example, the botulinum toxin type B toxin may beinactive. In one embodiment, the neurotoxin of the present invention isin an active state. In one embodiment, the neurotoxin is in an inactivestate. In one embodiment, a combination of active and inactiveneurotoxin is envisioned.

Kits

Also encompassed in the present invention is a kit comprising the BoNTor polypeptide disclosed herein (e.g., in the form of a pharmaceuticalcomposition). The kit may comprise one or more of the compositionsdescribed herein packaged in a vial. The kit may further comprise adelivery tool or device for the therapeutic administration of thecomposition, and/or instructions for therapeutic administration. The kitmay have all components therein packaged into a formed container.

Another aspect of the invention relates to a delivery tool or device foradministration of the pharmaceutical compositions described herein,pre-loaded with the pharmaceutical composition (e.g., for single use).Such devices may be a syringe or a microneedle device for delivery ofthe compositions. The syringe may be a single use syringe pre-loadedwith an effective amount of the composition. The microneedle device maycomprise one or more microneedles coated with the composition describedherein, such as is described in U.S. Patent Publication 2010/0196445,the contents of which are incorporated herein in their entirety.

Methods of Treatment

The present invention also includes methods for treating a conditiontypically treated with a neurotoxin (e.g, skeletal muscle conditions,smooth muscle conditions, glandular conditions, a neuromusculardisorder, an autonomic disorder, pain, or an aesthetic/cosmeticcondition). Such conditions are associated with unwanted neuronalactivity, as determined by the skilled practitioner. The methodcomprises the step of administering a therapeutically effective amountof a BoNT or polypeptide/chimeric molecule described herein (e.g. as apharmaceutical composition) to the appropriate location in the mammal toreduce the unwanted neuronal activity, to thereby treat the condition.Administration is by a route that contacts an effective amount of thecomposition to neurons exhibiting the unwanted activity.

Specific conditions envisioned for treatment by the methods discussedherein include, without limitation, spasmodic dysphonia, spasmodictorticollis, laryngeal dystonia, oromandibular dysphonia, lingualdystonia, cervical dystonia, focal hand dystonia, blepharospasm,strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focalspasticity and other voice disorders, spasmodic colitis, neurogenicbladder (i.e. all diseases involving urinary incontinence, such as e.g.,neurogenic detrusor overactivity or idiopathic overactive bladder),prostate cancer and other cancer forms, anismus, limb spasticity, tics,tremors, bruxism, anal fissure, achalasia, dysphagia and other muscletone disorders and other disorders characterized by involuntarymovements of muscle groups, lacrimation, hyperhydrosis, excessivesalivation, excessive gastrointestinal secretions as well as othersecretory disorders, pain from muscle spasms, neuropathic pain,inflammatory pain, headache pain, such as e.g. migraine, itch(pruritis), acne. In addition, the present invention can be used totreat dermatological or aesthetic/cosmetic conditions, for example,reduction of brow furrows, reduction of skin wrinkles. The presentinvention can also be used in the treatment of sports injuries.

In addition, the modified neurotoxin can be administered to treat otherneuromuscular disorders using well known techniques that are commonlyperformed with botulinum type A. For example, the present invention canbe used to treat pain, for example, headache pain, pain from musclespasms and various forms of inflammatory pain. For example, Aoki U.S.Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclose methods ofusing botulinum toxin type A for treating pain. The disclosure of thesetwo patents is incorporated in its entirety herein by reference.

Autonomic nervous system disorders can also be treated with a modifiedneurotoxin. For example, glandular malfunctioning is an autonomicnervous system disorder. Glandular malfunctioning includes excessivesweating and excessive salivation. Respiratory malfunctioning is anotherexample of an autonomic nervous system disorder. Respiratorymalfunctioning includes chronic obstructive pulmonary disease andasthma. Sanders et al. in U.S. Pat. No. 5,766,605 disclose methods fortreating the autonomic nervous system; for example, treating autonomicnervous system disorders such as excessive sweating, excessivesalivation, asthma, etc., using naturally existing botulinum toxins. Thedisclosure of Sander et al. is incorporated in its entirety by referenceherein. In one embodiment, substantially similar methods to that ofSanders et al. can be employed, but using a modified neurotoxin, totreat autonomic nervous system disorders such as the ones discussedabove. For example, a modified neurotoxin can be locally applied to thenasal cavity of the mammal in an amount sufficient to degeneratecholinergic neurons of the autonomic nervous system that control themucous secretion in the nasal cavity.

Pain that can be treated by a modified neurotoxin includes pain causedby muscle tension, or spasm, or pain that is not associated with musclespasm. For example, Binder in U.S. Pat. No. 5,714,468 discloses thatheadache caused by vascular disturbances, muscular tension, neuralgiaand neuropathy can be treated with a naturally occurring botulinumtoxin, for example Botulinum type A. The disclosures of Binder areincorporated in its entirety herein by reference. In one embodiment,substantially similar methods to that of Binder can be employed, butusing a modified neurotoxin, to treat headache, especially the onescaused by vascular disturbances, muscular tension, neuralgia andneuropathy. Pain caused by muscle spasm can also be treated by anadministration of a modified neurotoxin. For example, WO2006001676 (KangAhn) discloses methods of using a botulinum neurotoxin for treating kneejoint pain caused by saphenous nerve entrapment, WO2008059126 (ChristineFavre et al.) discloses methods of using a botulinum neurotoxin fortreating pain induced by chemotherapy, WO2008090287 (Christine Favre etal.) discloses methods of using a botulinum neurotoxin for treating paininduced by anti-HIV treatment, WO2009130600 (Christine Favre et al.)discloses methods of using a botulinum neurotoxin for treating painassociated with diabetic neuropathy, and WO2007144493 (Michel Auguet etal.) discloses methods of using a botulinum neurotoxin in combinationwith an opiate derivative for treating pain. The disclosures ofWO2006001676, WO2008059126, WO2008090287, WO2009130600, WO2007144493 areincorporated in its entirety herein by reference. Furthermore, amodified neurotoxin can be administered to a mammal to treat pain thatis not associated with a muscular disorder, such as spasm. In one broadembodiment, methods of the present invention to treat non-spasm relatedpain include central administration or peripheral administration of themodified neurotoxin.

An acute or chronic pain that is not associated with a muscle spasm canalso be alleviated with a local, peripheral administration of themodified neurotoxin to an actual or a perceived pain location on themammal. In one embodiment, the modified neurotoxin is administeredsubcutaneously at or near the location of pain, for example, at or neara cut. In some embodiments, the modified neurotoxin is administeredintramuscularly at or near the location of pain, for example, at or neara bruise location on the mammal. In some embodiments, the modifiedneurotoxin is injected directly into a joint of a mammal, for treatingor alleviating pain caused by arthritic conditions. Also, frequentrepeated injection or infusion of the modified neurotoxin to aperipheral pain location is within the scope of the present invention

Routes of administration for such methods are known in the art andeasily adapted to the methods described herein by the skilledpractitioner (e.g., see for example, Harrison's Principles of InternalMedicine (1998), edited by Anthony Fauci et al., 14th edition, publishedby McGraw Hill). By way of non-limiting example, the treatment of aneuromuscular disorder can comprise a step of locally administering aneffective amount of the molecule to a muscle or a group of muscles, thetreatment of an autonomic disorder can comprise a step of locallyadministering an effective of the molecule to a gland or glands, and thetreatment of pain can comprise a step of administering an effectiveamount of the molecule the site of the pain. In addition, the treatmentof pain can comprise a step of administering an effective amount of amodified neurotoxin to the spinal cord.

It is also envisioned that the modified H_(C) (e.g., B-H_(C)) describedherein can be utilized as a delivery tool to target neurons and othercell types that express human synaptotagmin II in humans. For example,the modified H_(C) linked to another bioactive molecule (e.g.,therapeutic agent) covalently or non-covalently to thereby form achimeric molecule described herein, can serve as the targeting vehicleto deliver the bioactive molecule to neurons and other cell types thatexpress human synaptotagmin II in humans by binding to human Syt Iand/or Syt II. As such, another aspect of the invention relates to theuse of a chimeric polypeptide molecule described herein to deliver thebioactive molecule to a neuron in a human subject. The second portion ofthe molecule can be a bioactive molecule such as a therapeutic agent(e.g., a polypeptide or drug). Linkage of the first and second portionsof the molecule can be covalent (e.g., in the form of a fusion protein)or non-covalent. Methods of such linkage are known in the art and canreadily be applied by the skilled practitioner. The chimeric polypeptidemolecule would be administered by a route that resulted in contacting ofthe polypeptide to the neurons expressing the receptor to which themodified B-H_(C) specifically binds (the target neuron), as describedherein.

Identifying Receptor Binding Activity

Another aspect of the invention relates to a method for identifying amodified BoNT receptor binding domain for its ability to bind to areceptor (e.g. a human Syt I or human Syt II, or human SV2). The methodutilizes the 2-hybrid assay system and utilizes fusions proteins made ofthe receptor and of the modified Hc respectively fused (expressed asfusions proteins) to the respective “bait” and “prey” subunits used inthe 2-hybrid assay (e.g., Gal 4 transcription activation systemutilizing an activation domain and a DNA binding domain). The 2-hybridassay is typically performed in yeast (S. cerevisiae), but similar assaysystems have been developed for use in other single celled organisms aswell such as in E. coli. Those systems are equally comparable. In oneembodiment, the bacterial adenylate cyclase 2-hybrid assay is used(Karimova et al., Proc Natl Acad Sci USA. May 12, 1998; 95(10):5752-5756, the contents of which are incorporated herein by reference).The system uses T18 and T25 as bait and prey, and can utilize E. coliBTH101 cells.

The modified Hc is expressed as a fusion protein with T18 (referred toas a first fusion protein) in the E. coli of the 2-hybrid assay. In themethod, the receptor (or a binding fragment thereof such as h-Syt IIa.a. 1-87) is co-expressed as a fusion protein with T25 (referred to asa second fusion protein) in the E. coli of the 2-hybrid assay. The assayutilizes a positive indicator of interaction between the respectivemolecules via their respective fusions. One possible positive indicatoris color development. Clonal E. coli colonies expressing both the firstand second fusion proteins are grown on solid media containing theappropriate selective and reporting media (e.g., ampicillin, kanamycin,X-gal, and IPTG) for an amount of time to allow for generation of apositive indication (e.g., color generation) from positive colonies.Binding of the modified binding domain to the receptor fragment willlead to the positive indication (e.g., expression of LacZ gene andresult in generation of a blue color in colonies grown on X-gal plates).

The colonies are analyzed for such positive indication (e.g., colordevelopment) when compared to appropriate controls (positive andnegative). The analyzing can be visually or by a non-human machine. Anappropriate negative control that fails to produce appreciable positiveindication (e.g., LacZ expression) is used (e.g, a clonal colony lackinga key component of the assay system such as having bait and prey thatare expressed without a fusion). An appropriate strong positive controlcan also be used. One example of a strong positive control for the humanSyt II receptor in the assay is a B1-Hc with a substitution mutation atE1191 (M/C/V or Q). A weakly positive control can also be used toidentify the strength of the binding. One example of a weakly positivecontrol for human Syt II receptor in the assay is a B1-Hc with asubstitution mutation at W1178 (Q/Y/A or S). Identification of thepositive indication (e.g., color development) indicates that themodified Hc binds the receptor. Identification of strong positiveindication such as strong color development (e.g. above a weaklypositive control) indicates the Hc binds the receptor with highaffinity.

The modified Hc used in the assay can be any modified Hc disclosedherein. By way of non-limiting example, the modified Hc may contain one,two or three substitution mutations such as those disclosed herein. Themodified Hc may be of any serotype/strain or subtype as disclosedherein.

The embodiments described here and in the following examples are forillustrative purposes only, and various modifications or changesapparent to those skilled in the art are included within the scope ofthe invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to describe the present invention,in connection with percentages means±1%.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising”). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

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Synaptotagmins I and II act as nerve cell receptors for botulinum    neurotoxin G. J Biol Chem 279, 30865-30870 (2004).-   13. Peng, L., et al. Botulinum neurotoxin D-C uses synaptotagmin    I/II as receptors and human synaptotagmin II is not an effective    receptor for type B, D-C, and G toxins. J Cell Sci (2012).-   14. Dong, M., et al. SV2 is the protein receptor for botulinum    neurotoxin A. Science 312, 592-596 (2006).-   15. Dong, M., et al. Glycosylated SV2A and SV2B mediate the entry of    botulinum neurotoxin E into neurons. Mol Biol Cell 19, 5226-5237    (2008).-   16. Mahrhold, S., Rummel, A., Bigalke, H., Davletov, B. & Binz, T.    The synaptic vesicle protein 2C mediates the uptake of botulinum    neurotoxin A into phrenic nerves. FEBS Lett 580, 2011-2014 (2006).-   17. Rummel, A., et al. Botulinum neurotoxins C, E and F bind    gangliosides via a conserved binding site prior to    stimulation-dependent uptake with botulinum neurotoxin F utilising    the three isoforms of SV2 as second receptor. J Neurochem 110,    1942-1954 (2009).-   18. Fu, Z., Chen, C., Barbieri, J. T., Kim, J. J. & Baldwin, M. R.    Glycosylated SV2 and gangliosides as dual receptors for botulinum    neurotoxin serotype F. Biochemistry 48, 5631-5641 (2009).-   19. Montecucco, C. How do tetanus and botulinum toxins bind to    neuronal membranes? TIBS, 314-317 (1986).-   20. Nishiki, T., et al. The high-affinity binding of Clostridium    botulinum type B neurotoxin to synaptotagmin II associated with    gangliosides GT1b/GD1a. FEBS Lett 378, 253-257 (1996).-   21. Pang, Z. P., et al. Synaptotagmin-2 is essential for survival    and contributes to Ca2+ triggering of neurotransmitter release in    central and neuromuscular synapses. J Neurosci 26, 13493-13504    (2006).-   22. Strotmeier, J., Willjes, G., Binz, T. & Rummel, A. Human    synaptotagmin-II is not a high affinity receptor for botulinum    neurotoxin B and G: increased therapeutic dosage and immunogenicity.    FEBS Lett 586, 310-313 (2012).-   23. Craxton, M. A manual collection of Syt, Esyt, Rph3a, Rph3al,    Doc2, and Dblc2 genes from 46 metazoan genomes—an open access    resource for neuroscience and evolutionary biology. BMC Genomics 11,    37 (2010).-   24. Brin, M. F., et al. Safety and efficacy of NeuroBloc (botulinum    toxin type B) in type A-resistant cervical dystonia. Neurology 53,    1431-1438 (1999).-   25. Pappert, E. J. & Germanson, T. Botulinum toxin type B vs. type A    in toxin-naive patients with cervical dystonia: Randomized,    double-blind, noninferiority trial. Mov Disord 23, 510-517 (2008).-   26. Wang, J., et al. Longer-acting and highly potent chimaeric    inhibitors of excessive exocytosis created with domains from    botulinum neurotoxin A and B. Biochem J 444, 59-67 (2012).-   27. Rummel, A., Mahrhold, S., Bigalke, H. & Binz, T. Exchange of the    H(CC) domain mediating double receptor recognition improves the    pharmacodynamic properties of botulinum neurotoxin. FEBS J 278,    4506-4515 (2011).-   28. Kozaki, S., et al. Characterization of Clostridium botulinum    type B neurotoxin associated with infant botulism in japan. Infect    Immun 66, 4811-4816 (1998).-   29. Ihara, H., et al. Sequence of the gene for Clostridium botulinum    type B neurotoxin associated with infant botulism, expression of the    C-terminal half of heavy chain and its binding activity. Biochim    Biophys Acta 1625, 19-26 (2003).-   30. Rummel, A., Mahrhold, S., Bigalke, H. & Binz, T. The HCC-domain    of botulinum neurotoxins A and B exhibits a singular ganglioside    binding site displaying serotype specific carbohydrate interaction.    Mol Microbiol 51, 631-643 (2004).-   31. Chai, Q., et al. Structural basis of cell surface receptor    recognition by botulinum neurotoxin B. Nature 444, 1096-1100 (2006).-   32. Jin, R., Rummel, A., Binz, T. & Brunger, A. T. Botulinum    neurotoxin B recognizes its protein receptor with high affinity and    specificity. Nature 444, 1092-1095 (2006).-   33. Arnon, S. S., et al. Botulinum toxin as a biological weapon:    medical and public health management. Jama 285, 1059-1070 (2001).-   34. Moriishi, K., et al. Mosaic structures of neurotoxins produced    from Clostridium botulinum types C and D organisms. Biochim Biophys    Acta 1307, 123-126 (1996).-   35. Hill, K. K., et al. Genetic diversity among Botulinum    Neurotoxin-producing clostridial strains. J Bacteriol 189, 818-832    (2007).-   36. Lalli, G., et al. Functional characterisation of tetanus and    botulinum neurotoxins binding domains. J Cell Sci 112 (Pt 16),    2715-2724 (1999).

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A botulinum neurotoxin (BoNT) polypeptide comprising:    -   a) a protease domain;    -   b) a protease cleavage site;    -   c) a translocation domain; and    -   d) a modified receptor binding domain of Clostridial botulinum        serotype B (B-H_(c)), comprising one or more substitution        mutations corresponding to substitution mutations in serotype B,        strain 1, selected from the group consisting of:        -   E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H,            W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and            combinations thereof.-   2. The BoNT polypeptide of paragraph 1, wherein the modified    (B-H_(c)) comprises one substitution mutation corresponding to a    substitution mutation in serotype B, strain 1, selected from the    group consisting of E1191C, E1191V, E1191L, E1191Y, S1199E, S1199H,    W1178Y, W1178Q, W1178A, W1178S, Y1183C, and Y1183P.-   3. The BoNT polypeptide of paragraph 2, wherein the modified    (B-H_(c)) comprises the substitution mutation corresponding to    E1191C in serotype B, strain 1.-   4. The BoNT polypeptide of paragraph 2, wherein the modified    (B-H_(c)) comprises the substitution mutation corresponding to    E1191V in serotype B, strain 1.-   5. The BoNT polypeptide of paragraph 1, wherein the modified    (B-H_(c)) comprises two substitution mutations.-   6. A botulinum neurotoxin (BoNT) polypeptide comprising:    -   a) a protease domain;    -   b) a protease cleavage site;    -   c) a translocation domain; and    -   d) a modified receptor binding domain of Clostridial botulinum        serotype B (B-H_(c)), comprising two or more substitution        mutations corresponding to substitution mutations in serotype B,        strain 1, wherein one of the substitution mutations is selected        from the group consisting of:        -   E1191Q, E1191M, E1191C, E1191V, E1191L, and E1191Y.-   7. The BoNT polypeptide of paragraph 6, wherein one other of the    substitution mutations corresponds to S1199W, S1199E, S1199H,    S1199Y, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, S1199F or    S1199L in serotype B, strain 1.-   8. The BoNT polypeptide of any one of paragraphs 5-7, wherein the    two substitution mutations correspond to E1191M and S1199W, E1191M    and W1178Q, E1191C and S1199W; E1191C and S1199Y, E1191C and W1178Q,    E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V    and W1178Q, in serotype B, strain 1.-   9. The BoNT polypeptide of any one of paragraphs 5-8, wherein the    two substitution mutations correspond to E1191M and S1199W in    serotype B, strain 1.-   10. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191M and W1178Q in serotype    B, strain 1.-   11. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191C and S1199W in serotype    B, strain 1.-   12. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191C and S1199Y in serotype    B, strain 1.-   13. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191C and W1178Q in serotype    B, strain 1.-   14. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191Q and S1199W in serotype    B, strain 1.-   15. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191V and S1199W in serotype    B, strain 1.-   16. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191V and S1199Y in serotype    B, strain 1.-   17. The BoNT polypeptide of one of paragraphs 5-8, wherein the two    substitution mutations correspond to E1191V and W1178Q in serotype    B, strain 1.-   18. The BoNT polypeptide of paragraph 6, wherein the modified    (B-H_(c)) comprises three substitution mutations.-   19. The BoNT polypeptide of paragraph 18, wherein the three    substitution mutations are at positions that correspond to E1191,    Y1183 and S1199 or to E1191, S1199 and W1178 of serotype B, strain    1.-   20. The BoNT polypeptide of paragraph 19, wherein the three    substitution mutations correspond to E1191M, S1199W and W1178Q of    serotype B, strain 1.-   21. A botulinum neurotoxin (BoNT) polypeptide comprising:    -   a) a protease domain;    -   b) a protease cleavage site;    -   c) a translocation domain; and    -   d) a modified receptor binding domain of Clostridial botulinum        serotype B (B-H_(c)), comprising a substitution mutation at a        position corresponding to S1199 or S1201 of serotype B, strain        1.-   22. The BoNT polypeptide of any one of paragraphs 1-21, wherein the    modified B-H_(c) is of strain 1.-   23. The BoNT polypeptide of any one of paragraphs 1-22 wherein the    protease domain, translocation domain, and protease cleavage site    are from serotype selected from the group consisting of A, B, C, D,    E, F, G, and combinations thereof.-   24. The BoNT polypeptide of paragraph 23, wherein the protease    domain, translocation domain, and protease cleavage site are from    serotype B, strain 1.-   25. The BoNT polypeptide of paragraph 23, wherein the protease    domain, translocation domain, and protease cleavage site are from    serotype A, strain 1.-   26. A polypeptide comprising a modified receptor binding domain of    Clostridial botulinum serotype B (B-H_(c)) comprising one or more    substitution mutations corresponding to substitution mutations in    serotype B, strain 1, selected from the group consisting of:    -   E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y,        W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.-   27. The polypeptide of paragraph 26, wherein the modified (B-H_(c))    comprises two substitution mutations.-   28. A polypeptide comprising a modified receptor binding domain of    Clostridial botulinum serotype B (B-H_(c)) comprising two or more    substitution mutations corresponding to substitution mutations in    serotype B, strain 1, wherein one of the substitution mutations is    selected from the group consisting of E1191Q, E1191M, E1191C,    E1191V, E1191L, and E1191Y.-   29. The polypeptide of paragraph 26, wherein one of the substitution    mutations corresponds to S1199W, S1199E, S1199H, S1199Y, W1178Y,    W1178Q, W1178A, W1178S, Y1183C, Y1183P, S1199F or S1199L in serotype    B, strain 1.-   30. The polypeptide of any one of paragraphs 27-29, wherein the two    substitution mutations correspond to E1191M and S1199W, E1191M and    W1178Q, E1191C and S1199W; E1191C and S1199Y, E1191C and W1178Q,    E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V    and W1178Q, in serotype B, strain 1.-   31. The polypeptide of any one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191M and S1199W in serotype    B, strain 1.-   32. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191M and W1178Q in serotype    B, strain 1.-   33. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191C and S1199W in serotype    B, strain 1.-   34. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191C and S1199Y in serotype    B, strain 1.-   35. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191C and W1178Q in serotype    B, strain 1.-   36. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191Q and S1199W in serotype    B, strain 1.-   37. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191V and S1199W in serotype    B, strain 1.-   38. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191V and S1199Y in serotype    B, strain 1.-   39. The polypeptide of one of paragraphs 27-30, wherein the two    substitution mutations correspond to E1191V and W1178Q in serotype    B, strain 1.-   40. The polypeptide of paragraph 30, wherein the modified (B-H_(c))    comprises three substitution mutations.-   41. The polypeptide of paragraph 40, wherein the three substitution    mutations are at positions that correspond to E1191, Y1183 and S1199    or to E1191, S1199 and W1178 of serotype B, strain 1.-   42. The polypeptide of paragraph 41, wherein the three substitution    mutations correspond to E1191M, S1199W and W1178Q of serotype B,    strain 1.-   43. The polypeptide of any one of paragraphs 26-42, wherein the    modified B-H_(c) is of strain 1.-   44. A chimeric molecule comprising a first portion that is a    modified receptor binding domain of Clostridial botulinum serotype B    (B-H_(c)) linked to a second portion, wherein the modified B-H_(c)    comprises one or more substitution mutations selected from the group    consisting of:    -   E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y,        W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.-   45. The chimeric molecule of paragraph 44, wherein the modified    B-H_(c) comprises two substitution mutations.-   46. A chimeric molecule comprising a first portion that is a    modified receptor binding domain of Clostridial botulinum serotype B    (B-H_(c)) linked to a second portion, wherein the modified B-H_(C)    comprises two or more substitution mutations corresponding to    substitution mutations in serotype B, strain 1, wherein one of the    substitution mutations is selected from the group consisting of:    -   E1191Q, E1191M, E1191C, E1191V, E1191L, and E1191Y.-   47. The chimeric molecule of paragraph 46, wherein one of the    substitution mutations corresponds to S1199W, S1199E, S1199H,    S1199Y, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, S1199F or    S1199L in serotype B, strain 1.-   48. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191M and S1199W,    E1191M and W1178Q, E1191C and S1199W; E1191C and S1199Y, E1191C and    W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or    E1191V and W1178Q, in serotype B, strain 1.-   49. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191M and S1199W in    serotype B, strain 1.-   50. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191M and W1178Q in    serotype B, strain 1.-   51. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191C and S1199W in    serotype B, strain 1.-   52. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191C and S1199Y in    serotype B, strain 1.-   53. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191C and W1178Q in    serotype B, strain 1.-   54. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191Q and S1199W in    serotype B, strain 1.-   55. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191V and S1199W in    serotype B, strain 1.-   56. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191V and S1199Y in    serotype B, strain 1.-   57. The chimeric molecule of any one of paragraphs 45-47, wherein    the two substitution mutations correspond to E1191V and W1178Q in    serotype B, strain 1.-   58. The chimeric molecule of one of paragraph 45 or 46, wherein the    modified (B-H_(c)) comprises three substitution mutations.-   59. The chimeric molecule of paragraph 58, wherein the three    substitution mutations are at positions that correspond to E1191,    Y1183 and S1199 or to E1191, S1199 and W1178 of serotype B, strain    1.-   60. The chimeric molecule of paragraph 59, wherein the three    substitution mutations correspond to E1191M, S1199W and W1178Q of    serotype B, strain 1.-   61. The chimeric molecule of any one of paragraphs 44-60, wherein    the modified B-H_(c) is of strain 1.-   62. The chimeric molecule of any one of paragraphs 44-61, wherein    the first portion and the second portion are linked covalently.-   63. The chimeric molecule of any one of paragraphs 46-61, wherein    the first portion and the second portion are linked non-covalently.-   64. The chimeric molecule of any one of paragraphs 44-61 wherein the    second portion is selected from the group consisting of a small    molecule, a nucleic acid, a short polypeptide and a protein.-   65. The chimeric molecule of paragraph 64, wherein the second    portion is a bioactive molecule.-   66. The chimeric molecule of paragraph 64 or 65, wherein the second    portion is a therapeutic polypeptide or non-polypeptide drug.-   67. The BoNT polypeptide, polypeptide or chimeric molecule of any    one of paragraphs 1-66 that exhibits significantly enhanced binding    of the modified B-H_(c) to human SytII and/or significantly reduced    binding of the modified B-H_(c) to human Syt I as compared to an    identical molecule lacking the substitution mutation(s).-   68. The BoNT polypeptide, polypeptide or chimeric molecule of any    one of paragraphs 1-66 wherein the substitution mutation produces    significantly enhanced binding to human SytII and/or significantly    enhanced binding to human Syt I as compared to an identical molecule    lacking the substitution mutation(s).-   69. A nucleic acid comprising a nucleotide sequence that encodes the    polypeptide or chimeric molecule of any one of paragraphs 1-68.-   70. A nucleic acid vector comprising the nucleic acid of paragraph    69.-   71. A cell comprising the nucleic acid vector of paragraph 70 or the    nucleic acid of paragraph 69.-   72. A cell expressing the polypeptide or chimeric molecule of any    one of paragraphs 1-68-   73. A pharmaceutical composition comprising the botulinum neurotoxin    (BoNT) polypeptide of any one of paragraphs 1-25, 67 or 68, or the    chimeric molecule of any one of paragraphs 44-66, or the nucleic    acid vector of paragraph 70 or the nucleic acid of paragraph 69.-   74. The pharmaceutical composition of paragraph 73, further    comprising a pharmaceutically acceptable excipient.-   75. A kit comprising a pharmaceutical composition of paragraph 73 or    74 and directions for therapeutic administration of the    pharmaceutical composition.-   76. A method to produce a botulinum neurotoxin (BoNT) polypeptide,    the method comprising the steps of culturing the cell of paragraph    72 under conditions wherein said BoNT polypeptide is produced.-   77. The method of paragraph 76 further comprising one or more of the    following steps:    -   recovering the BoNT polypeptide from the culture,    -   purifying the BoNT polypeptide,    -   activating the BoNT polypeptide, and/or    -   formulating the BoNT polypeptide.-   78. A method for treating a condition associated with unwanted    neuronal activity comprising administering a therapeutically    effective amount of the BoNT polypeptide of any one of paragraphs    1-25, 67 or 68 to a subject to thereby contact one or more neurons    exhibiting unwanted neuronal activity, to thereby treat the    condition.-   79. The method of paragraph 78, wherein the condition is selected    from the group consisting of, spasmodic dysphonia, spasmodic    torticollis, laryngeal dystonia, oromandibular dysphonia, lingual    dystonia, cervical dystonia, focal hand dystonia, blepharospasm,    strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal    spasticity and other voice disorders, spasmodic colitis, neurogenic    bladder, anismus, limb spasticity, tics, tremors, bruxism, anal    fissure, achalasia, dysphagia and other muscle tone disorders and    other disorders characterized by involuntary movements of muscle    groups, lacrimation, hyperhydrosis, excessive salivation, excessive    gastrointestinal secretions, secretory disorders, pain from muscle    spasms, headache pain, migraine, and dermatological or    aesthetic/cosmetic conditions.-   80. The botulinum neurotoxin (BoNT) polypeptide of any one of    paragraphs 1-25, 67 or 68, the pharmaceutical composition of one of    paragraphs 73 or 74 or the chimeric molecule of any one of    paragraphs 44-66, or the polypeptide of any one of paragraphs 26-43,    for use in medicine.-   81. The botulinum neurotoxin (BoNT) polypeptide of any one of    paragraphs 1-25, 67 or 68, the pharmaceutical composition of one of    paragraphs 73 or 74, or the chimeric molecule of any one of    paragraphs 44-66, or the polypeptide of any one of paragraphs 67-43,    for use in treating a condition associated with unwanted neuronal    activity.-   82. A method for identifying a modified receptor binding domain of    botulinum neurotoxin for binding to a receptor comprising;    -   a) expressing the modified receptor binding domain as a first        fusion protein with T18 subunit of bacterial adenylate cyclase        in a 2-hybrid assay, and expressing the receptor as a second        fusion protein with T25 subunit of bacterial adenylate cyclase        in the 2-hybrid assay;    -   b) analyzing a clonal E. coli colony that expresses both the        first and second fusion protein for the presence of a positive        indication above a negative control; and    -   c) identifying the modified receptor binding domain expressed by        the colony exhibiting the positive indication as binding to the        receptor.-   83. The method of paragraph 82 wherein the positive indication is    color development.-   84. The method of any one of paragraphs 82-83, further comprising    the step of analyzing the colony for the positive indication against    a weakly positive control, and further identifying the modified    receptor binding domain expressed by the colony exhibiting above the    weakly positive control as binding to the receptor with high    affinity.-   85. The method of paragraph 82-84 that is performed with a library    of modified receptor binding domains each expressed within    respective colonies.-   86. The method of any one of paragraphs 82-85 wherein the receptor    is human.-   87. The method of any one of paragraphs 82-86 wherein the modified    receptor binding domain comprises one or more substitution    mutations, wherein one of the substitution mutations corresponds to    a mutation in serotype B, strain 1, selected from the group    consisting of: E1191M, E1191 Q, E1191C, E1191V, E1191L, E1191Y,    S1199W, S1199E, S1199H, S1199F, S1199L W1178Y, W1178Q, W1178A,    W1178S, Y1183C, Y1183P and S1199Y.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES

Identification of Single Mutations that Enhance BoNT/B Binding to h-SytII

The co-crystal structure of BoNT/B bound to rat Syt II luminal domainhas been solved by two studies in 2006^(25,26). The structuralinformation provided a sound basis for us to focus on limited numbers ofresidues within the binding interface for rational design mutagenesisstudies. The conserved phenylalanine at position 54 forms multiplehydrophobic contacts with BoNT/B. Because leucine (in humans) is alsohydrophobic, disruption of BoNT/B binding is likely due to size/shapedifferences between phenylalanine and leucine. The key was to identifypossible changes in BoNT/B H_(c) region that may accommodate the changefrom phenylalanine to leucine.

All residues in BoNT/B that contribute to interactions between BoNT/Band Syt II were examined. These residues were well-defined by theBoNT/B-Syt II co-crystal structure, including K1113, D1115, S1116,P1117, V1118, W1178, Y1181, Y1183, E1191, K1192, F1194, A1196, P1197,S1199, S1201, E1203, F1204, E1245, and Y1256 (FIG. 3), a total of 19positions. The strategy was to first systematically replace the residuesat each of these 19 positions with all other 19 possible amino acids andthen test binding of these mutated BoNT/B-H_(C) to h-Syt II. Therefore,a total of 19×19=361 single point mutations needed to be generated andtested.

A bacterial adenylate cyclase two hybrid system (BACTH) was used togenerate these 361 mutations and test their binding to h-Syt II asdescribed in FIG. 4. Briefly, wild type (WT) BoNT/B-H_(C) was subclonedinto a vector in frame with the split fragment (T18) of the bacterialadenylate cyclase. This T18-BoNT/B-H_(C) fusion construct was amplifiedby PCR with primers harboring random tri-nucleotides (NNN) at theselected position in BoNT/B-H_(C) (FIG. 4A). This generated a pool ofconstructs that encode all 20 different amino acids at the select site.This pool of constructs were then co-transformed into bacteria (E. colistrain BTH101), together with a construct that expresses h-Syt II (1-87)fused with the other half of the split bacterial adenylate cyclase(T25). Binding of a mutant BoNT/B-H_(C) to h-Syt II brought T18 and T25together and recovered the activity of adenylate cyclase, which lead toexpression of lacZ gene and results in blue colonies on X-gel plates(FIG. 4B). The specific mutations introduced into BoNT/B-H_(C) wereidentified by extracting and sequencing the constructs from these bluecolonies.

Using this BACTH method, all 19 selected sites were screened inBoNT/B-H_(C). The numbers of total colonies and blue colonies for eachsite are listed in FIG. 5A. Greater than 380 total colonies were countedfor each position. The total colony number determined the possibility ofcovering all 20 amino acids at the selected mutation site. This wascalculated by Clark-Carbon equation: P=1−(1−f)^(N), where f reflects thenumber of possible residues (f=1/20 here as there are 20 different aminoacids), and N is the total number of colonies. With a minimal number of380 colonies, the probability of covering all 20 amino acids at aposition is 99.8%. Therefore, it is likely that all 20 possible residueswere covered once the number of colonies on the plates was over 380.

As shown in FIG. 5A, four positions were found to result in bluecolonies, including E1191 (22.7%), W11178 (7.8%), Y1183 (4.0%), andS1199 (5.1%). Among these four positions, E1191 had the highest levelsof blue colonies. In addition, the blue colonies from E1191 also showeddeeper blue color as compared to the other three sites, which suggestedthat the interaction between BoNT/B-H_(C) and h-Syt II could be strongerwith E1191 mutations. Plasmids were extracted and sequenced from theseblue colonies. The identified residues at each site are listed in FIG.5B: E1191M/C/V/Q/L/Y, Y1183/C/P, S1199W/E/Y/H, W1178Y/Q/A/S.

The interactions between human Syt II and those identified mutants werefurther verified by measuring the levels of β-galactosidase produced inbacteria, which was proportional to the total level of adenylate cyclaseactivity reconstituted by interactions between T18-BoNT/B-H_(C) andT25-h-Syt II. Four mutations at E1191 sites, E1191M/C/V/Q, showed thestrongest β-galactosidase activity among all mutations tested (FIG. 6A),suggesting that replacing E1191 with one of these four residuessignificantly enhanced binding of T18-BoNT/B-H_(C) to T25-h-Syt II.

Binding of mutant BoNT/B-H_(C) to h-Syt II was further analyzed usingpull-down assay as an alternative approach (FIG. 6B). Briefly,GST-tagged mouse Syt II luminal domain (m-Syt II) and human Syt IIluminal domain (h-Syt II) were immobilized on GST beads and were used topull down mutant BoNT/B-H_(C) expressed in bacteria. Binding ofBoNT/B-H_(C) to GST-tagged Syt II was detected via immunoblot analysis,detecting the HA tag fused to BoNT/B-H_(C). As shown in FIG. 6B,E1191M/C/V/Q resulted in significant levels of binding to h-Syt II,which was consistent with the finding that these four mutations alsodisplayed the strongest β-galactosidase activity (FIG. 6A). Together,these results indicated that E1191M/C/V/Q are four primary mutationsthat gain the ability to bind h-Syt II robustly.

Combinational Mutations in HCB Further Enhanced its Binding to h-Syt II

Whether binding of BoNT/B-H_(C) E1191M/C/V/Q to h-Syt II can be furtherenhanced by including a secondary mutation at a different site was thenexplored. The 1183, 1199, and 1178 sites were focused on as thecandidates for the secondary mutation sites, as these are the only threesites that also resulted in blue colonies (FIG. 5). Using E1191M as theprimary mutations, double mutations were generated combining E1191M withall other 10 residue changes identified in BACTH screening at 1183,1199, and 1178 sites as indicated in FIG. 5B. These 10 double mutationswere analyzed for their ability to bind h-Syt II in pull-down assays asindicated in FIG. 7A. Three of them, E1191M/S1199W, E1191M/S1199Y, andE1191M/W1178Q, resulted in significant binding to h-Syt II (FIG. 7A).These results suggested that including S1199W/Y and W1178Q as thesecondary mutation site would further enhance binding of BoNT/B-H_(C)E1191M/C/V/Q to h-Syt II. Together, these data indicated that there arefour choices of primary mutation at E1191 site (M/C/V/Q) and threechoice of secondary mutation sites (S1199W/Y and W1178Q) (FIG. 7B), anda combination of a primary mutation with a secondary mutation canfurther enhance binding to h-Syt II.

Combining E1191M with Y1183C/P actually reduced binding to h-Syt II(FIG. 7A). The Syt II binding interface of BoNT/B is composed of twohydrophobic pouches as previously reported. E1191 and Y1183 are locatedin the same pouch, while S1199 and W1178 are located in another. BecauseE1191 is spatially close to Y1183, a potential structural conflict mayoccur when mutating both of them, which may explain why double mutationsat these two positions reduced binding to h-Syt II.

Determination and comparison of binding affinities between mutatedBoNT/B-H_(C) to h-Syt II quantitatively was pursued, aiming to selectthe double mutations with the best binding affinity. The bindingaffinity (K_(D)) was determined using a well-established bio-layerinterferometry assay (FIG. 8A). Briefly, GST tagged Syt proteins wereimmobilized onto a probe. The probe was first exposed to purifiedBoNT/B-H_(C) at different concentrations (association phase, FIG. 8A),followed by washing steps (dissociation phase, FIG. 8A). Binding ofBoNT/B-H_(C) to GST tagged Syt increases the total molecular weight/sizeon the probe, which results in a shift in light reflection at the probethat can be detected and analyzed. Binding parameters such asassociation constant (K_(on)), dissociation constant (K_(off)), andapparent binding affinity (K_(D)) can be calculated from the associationand dissociation curve detected as indicated in FIG. 8A.

Using this assay, all combinations of four primary mutations(E1191M/C/Q/V) were systematically characterized with three secondarymutations (S1199W/Y, W1178Q). In addition, a triple mutation,E1191M/S1199W/W1178Q, was also generated and analyzed. Binding of WTBoNT/B-H_(C) to mouse Syt II (m-Syt II) was measured as a positivecontrol, which showed a binding K_(D) at 0.13 μM (FIG. 8B). As expected,binding of WT BoNT/B-H_(C) to h-Syt II was too weak to be reliablydetermined, with an estimated K_(D) over the detection limit (>20 μM). Asingle primary mutation E1191M yielded a binding K_(D) at 6.7 μM, asignificant improvement over WT BoNT/B-H_(C). Double mutations thatcombined a primary mutation site with a secondary mutation sites furtherimproved the binding affinity to as high as 0.59 μM (E1191V/S1199Y). Asexpected, the majority of double mutations improved the binding affinityto h-Syt II, with K_(D) between 0.59 μM to 4.3 μM. E1191Q/W1178Q was theonly one that did not bind to h-Syt II. The reason is not known, but itis possible that this double mutation may have induced unexpectedconformational changes of the protein. The triple mutationE1191M/S1199W/W1178Q showed almost the same binding affinity as thedouble mutation E1191M/S1199W, suggesting that adding the third mutationsite may not further improve the binding affinity. Together, these dataconfirmed that all double mutations between E1191M/C/Q/V and S1199W/Y,W1178Q, with the exception of E11191Q/W1178Q, resulted in mutantBoNT/B-H_(C) that can bind to h-Syt II robustly.

HCB Mutants Showed Enhanced Binding to h-Syt I

In addition to Syt II, Syt I also functions as a receptor for BoNT/B. Inorder to achieve the highest possible binding to human neurons, themodified BoNT/B mutants should not affect binding to human Syt I.Ideally, they may even increase binding to Syt I. Indeed, it was foundthat E1191M significantly enhanced binding of BoNT/B-H_(C) to h-Syt I,as robust binding could be detected without the presence of the lipidco-receptor gangliosides (FIG. 9A). The binding affinity betweenselected double mutations and h-Syt I was measured using bio-layerinterferometry assay. As shown in FIG. 9B, C, double mutationsE1191M/S1199Y (B-H_(C) MY) and E1191V/S1199Y (B-H_(C) VY) displayedK_(D) at 2.9 μM and 5.82 μM, respectively, whereas the WT BoNT/B-H_(C)binding to h-Syt I was too weak to be reliably determined, withestimated K_(D) over the detection limit (>20 μM).

H_(C)B_(MY) Binds to h-Syt II on Neuronal Surfaces

We next examined whether H_(C)B_(MY) mutant may bind to h-Syt II onphysiologically relevant neuronal surfaces. To this end, we utilizedcultured rat cortical neurons as a neuron model, which expresses Syt Ibut not Syt II (Dong et al, The Journal of cell biology 179(7):1511-1522(2007)). Thus, knocking down Syt I generated neurons with no endogenousreceptors. Expressing full-length h-Syt II in these Syt I KD neuronscreated “humanized” neurons with only h-Syt II as the toxin receptor aswe previously described (Peng et al., J Cell Sci. 125: 3233-42(2012)).As expected, WT H_(C)B bound strongly to rat neurons and the binding wasabolished after knocking down endogenous Syt I. Expression offull-length m-Syt II, but not h-Syt II nor a m-Syt II containing F54Lmutation, restored binding of WT H_(C)B (FIG. 10A). In contrast,H_(C)B_(MY) showed robust binding to neurons that express m-Syt II,h-Syt II, or m-Syt II (F54L), demonstrating that H_(C)B_(MY) gain theenhanced ability to bind h-Syt II on neuronal surfaces (FIG. 10B).

BoNT/B Mutant Displayed Enhanced Efficacy to Block Neurotransmission inHumanized Neurons

To address key question whether enhanced binding to h-Syt II translatesto improved efficacy at functional levels in neurons, we producedfull-length WT BoNT/B and the mutant toxin containing E1191M/S1199Ypoint mutations (BoNT/B_(MY)) recombinantly in E. coli. Humanizedneurons were exposed to a gradient of WT or BoNT/B_(MY) toxins. Cleavageof VAMP2 was examined by immunoblotting analysis. As shown in FIG. 11A,more VAMP2 was cleaved in neurons exposed to BoNT/B_(MY) compared toneurons exposed to WT BoNT/B at each toxin concentration tested,indicating that BoNT/B_(MY) targeted and entered neurons moreefficiently than WT toxin.

We next monitored neurotransmitter release by recording miniatureinhibitory postsynaptic currents (mIPSCs) using whole-cell patch-clamprecording. The frequency of mIPSCs reflects the activity ofneurotransmitter release in a population of neurons. Entry of BoNT/Binto presynaptic terminals blocks release of neurotransmitter, thusreducing frequency of mIPSCs (FIG. 11B). Humanized neurons were exposedto a gradient of WT BoNT/B or BoNT/B_(MY). As shown in FIG. 11C,BoNT/B_(MY) showed a greatly enhanced potency, with a half maximuminhibitory concentration (IC₅₀)˜11-fold lower than WT toxin: BoNT/B_(MY)can achieve the same level of blockage on neurotransmitter release with11-fold lower toxin concentrations compared to WT toxin. These datademonstrated that enhanced binding to human receptors resulted inincreased efficacy of toxin at functional levels in neurons.

The BACTH method was used to screen all possible single mutations at all19 key residues in BoNT/B-H_(C) that form the binding pocket for Syt II.Four positions were identified that can be mutated to increase thebinding affinity to h-Syt II, with E1191 site as the primary site, andS1199, W1178, and Y1183 as the secondary mutation sites. Doublemutations combining the primary and secondary mutation sites, werecreated and tested, and showed that combining E1191M/C/V/Q with S1199Y/Wor W1178Q yield double mutations with strong binding affinity to h-SytII and h-Syt I.

DISCUSSION

By combining rationale design based on available co-crystal structure ofthe BoNT/B-Syt II complex with the BACTH method that saturates allpossible single point mutations at each of the selected target residues,a series of point mutations in BoNT/B was identified that can bind toh-Syt II. These point mutations were further examined in combinations,revealing double mutants that gained high-affinity binding to h-Syt II.Importantly, full-length BoNT/B containing the designed mutation showed˜11-fold higher potency than WT BoNT/B on humanized neurons,demonstrating that enhanced binding to toxin receptors translates tohigher potency at functional levels in neurons.

Materials and Methods

Materials and Constructs.

The following antibodies were purchased from indicated vendors: SynapsinI (Clone 46.1, Synaptic Systems), VAMP2 (Clone 69.1, Synaptic Systems),HA (16B12, Covance), and β-tubulin III (ab18207, Abcam). Bovine mixedbrain gangliosides were purchased from Matreya LLC (Pleasant Gap, Pa.)and were reconstituted in Tris-buffered saline (TBS: 20 mM Tris, 150 mMNaCl) as previously described (Peng et al., PLoS pathogens7(3):e1002008(2011)). The cDNA encoding H_(C)B (residue 857-1291,Genbank: ACA46990.1) was codon optimized for E. coli expression andsynthesized by Genscript Inc. (New Brunswick, N.J.). The following cDNAswere generously provided by indicated groups: rat Syt I (T. C. Sudhof,Palo Alto, Calif.), mouse Syt II (M. Fukuda, Ibaraki, Japan), human SytI (R. B. Sutton, Lubbock, Tex.). DNA encoding H_(C)B was subcloned intopET28a vector, with both a His6 tag (SEQ ID NO: 13) and a HA tag(YPYDVPDYA (SEQ ID NO: 12)) fused to its N-terminus. Mutations in H_(C)Bwere generated via PCR using Site-directed Mutagenesis Kit (AgilentTechnologies, CA). GST tagged Syt I/II fragments and Syt II F54L mutantwere described previously (Dong M, et al. The Journal of cell biology162(7):1293-1303(2003); Peng et al., J Cell Sci. 125: 3233-42 (2012);Dong M, et al. Science 312(5773):592-596 (2006)).

BACTH (Bacterial Adenylate Cyclase Two-Hybrid Assay):

The BACTH assay was performed according to the manufacturer'sinstruction (Euromedex). Two compatible plasmids, pUT18C and pKT25 wereselected for the screen. H-Syt II luminal domain (residues 1-80) wascloned into pKT25 to generate pKT25-h-Syt II. HCB was cloned in pUT18Cfor producing T18-HCB. HCB mutant libraries were created with primerscontaining random nucleotide triplets (NNN) at designated positions.Each library was co-transformed with the pKT25-h-Syt II plasmid into E.coli indicator strain BTH101 by electroporation and screened on LB agarplates containing 100 μg/ml Ampicillin, 50 μg/ml Kanamycin, 0.5 mM IPTG,and 40 μg/ml X-Gal. The plates were incubated at 30° C. for 64 hours.Plasmids were extracted from blue colonies and sequenced. The totalcolony number determined the possibility of covering all 20 amino acidsat the selected mutation site. This was calculated by Clark-Carbonequation: P=1−(1−f)^(N), where f reflects the number of possibleresidues (f=1/20 here as there are 20 different amino acids), and N isthe total number of colonies. With a minimal number of 380 colonies inthe assays, the probability of covering all 20 amino acids at eachposition is >99.8%.

β-Galactosidase Assay:

E. coli BTH101 cells with expressed proteins of interest were inoculatedinto liquid LB medium containing ampicillin, kanamycin, and IPTG (0.5mM). The culture was grown overnight at 37° C. to reach the stationaryphase. The OD₆₀₀ of the culture grown overnight was recorded beforeharvesting. One milliliter of the culture grown overnight wascentrifuged, and cell pellets were washed twice with PBS and suspendedin an equal volume of Z buffer (60 mM Na2HPO4, 40 mM Na₂HPO₄, 10 mM KCl,1 mM MgSO₄, and 20 mM dithiothreitol [DTT]). One hundred microliters ofresuspended bacterial cells was diluted in 1 ml of Z buffer (dilutionfactor [DF]=10). Afterwards, 100 ml of chloroform and 50 ml of 0.1% SDSwere added and mixed well to permeabilize the cells. Two hundred fiftymicroliters of the mixture was then transferred into a new Microfugetube and brought to 28° C., 50 ml of pre-warmedo-nitrophenyl-β-galactoside (4 mg/ml in Z buffer) was added, and themixture was incubated at 28° C. until a yellow color developed. Thereaction was stopped by addition of 200 ml of 1 M Na₂CO₃. The A₄₂₀ andthe precise time period of the reaction in minutes (T) were recorded.β-galactosidase activity was defined as (1000×A₄₂₀×DF)/(T×OD₆₀₀) inMiller units.

Protein Expression and Purification:

WT and mutants of BoNT/B-H_(C) were expressed as His₆ tagged recombinantproteins (“His₆” disclosed as SEQ ID NO: 13) in E. coli. Syt I/IIfragments and mutants were expressed as GST tagged recombinant proteinsin E. coli. Both GST-fusion and His₆-fusion proteins (“His₆” disclosedas SEQ ID NO: 13) were purified as previously described⁹, with theinduction temperature at 20° C. overnight with 0.25 mM IPTG.

GST Pull-Down Assays:

Two types of pull-down assays were carried out. The first series wereused to screen binding of mutant BoNT/B-H_(C) to GST-tagged mouse Syt II(m-Syt II) and a mutant mouse Syt II (F54L) that mimicking human Syt IIsequence (designated as h-Syt II). Briefly, 6 ml of E. coli expressingBoNT/B H_(c) were spin down, re-suspended in 800 μl TBS, sonicated, andthen incubated with 2% Triton X-100 for 1 hr at 4° C. Samples were thenspun down at maximal speed for 15 min in a microcentrifuge at 4° C. Thesupernatants were collected and were used for pull-down assays byincubating with 10 μg of Syt proteins immobilized onglutathione-Sepharose beads (GE bioscience, Piscataway, N.J.) at 4° C.for 1 hr. Samples were washed three times in washing buffer (TBS with0.5% Triton X-100), and analyzed by immunoblotting BoNT/B-H_(C) usingthe anti-HA antibody. For mutants with enhanced binding to h-Syt II,further pull-down assays were carried out by purifying theseBoNT/B-H_(C) mutants as His6 tagged proteins (“His6” disclosed as SEQ IDNO: 13) as described previously⁹. Pull-down assays were then carried outusing immobilized Syt fragments in 100 μl TBS buffer plus 0.5% TritonX-100, with or without gangliosides (60 μg/ml), for 1 hr at 4° C. Beadswere washed three times using TBS buffer plus 0.5% Triton X-100. Tenpercent of bound materials were subjected to SDS-PAGE followedimmunoblot analysis.

Biolayer Interferometry Assay.

The binding affinities between H_(C)B variants and Syt I/Syt II weremeasured by BLI assay with the Blitz system (ForteBio). Briefly, theGST-tagged Syt I or Syt II (20 μg/ml) were immobilized onto Dip andRead™ Anti-GST Biosensors (ForteBio) and balanced with PBS buffer. Thebiosensors were then exposed to series concentrations of H_(C)B,followed by washing with PBS. Binding affinities (K_(D)) were calculatedusing the Blitz system software following manufacture's instruction(ForteBio).

Neuron Culture, Lentivirus, and Toxin Binding/Entry Assay.

Rat cortical neurons were prepared from E18-19 embryos as describedpreviously (Peng et al. PLoS pathogens 7(3):e1002008 (2011)). Constructsfor Syt I KD, mSyt II, and h-Syt II expression in neurons was previouslydescribed (Peng et al., J Cell Sci. 125: 3233-42(2012)). Lentiviruseswere added to neuron cultures at DIV5 (days in vitro), and toxinbinding/entry experiments were carried out on DIV12-14. Toxins werediluted in high K⁺ buffer (87 mM NaCl, 56 mM KCl, 1.5 mM KH₂PO₄, 8 mMNa₂HPO₄, 0.5 mM MgCl₂, and 1 mM CaCl) and pre-warmed to 37° C. Neuronswere exposed to above toxin-containing buffers for 5 minutes at 37° C.followed by washing with PBS. These neurons were either subjected toimmunostaining analysis, or incubated in toxin-free medium foradditional 24 hours, followed by immunoblotting analysis.

mIPSC Recording.

Whole-cell patch-clamp recordings were made from DIV 14-18 culturedcortical neurons (DIV 14-18). The pipette solution contained (in mM):135 CsCl, 10 HEPES, 1 EGTA, 1 Na-GTP, 4 Mg-ATP and 10 QX-314 (pH 7.4,adjusted with CsOH). The resistance of pipettes filled withintracellular solution varied between 4 and 5 MΩ. After formation of thewhole-cell configuration and equilibration of the intracellular pipettesolution, the series resistance was adjusted to 10 MΩ. Synaptic currentswere monitored with an EPC-10/2 amplifier (HEKA) at −70 mV holdingpotential. The bath solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl,1 MgCl2, 10 HEPES, 10 glucose (pH 7.4, adjusted with NaOH). Spontaneousinhibitory postsynaptic currents (sIPSCs) and evoked inhibitorypostsynaptic currents (eIPSCs) were pharmacologically inhibited byadding AMPA and NMDA receptor blockers CNQX and APV to the extracellularbath solution. Spontaneous miniature inhibitory postsynaptic currents(mIPSCs) were monitored in the presence of tetrodotoxin (TTX) to blockaction potentials. Data were analyzed using Clampfit 10 (MolecularDevices), Origin8 software (Mocrocal Inc.), MiniAnalysis software(Synaptosoft), and Igor (Wavemetrics). Statistical analysis wasperformed with Student's t-test (*P<0.01). All data shown aremeans±S.E.M.s.

CONCLUSION

The above-presented results indicate new methods and compositions toimprove binding of BoNT/B to its human receptors, and provide a way tocreate new generations of therapeutic BoNTs, by utilizing the modifiedreceptor binding domain created in the present invention, with improvedefficacy and specificity to target human neurons than the currentlyutilized WT BoNTs.

These efforts show that modifying the protein sequence of BoNT/B-H_(C)can create new versions that can bind human Syt II with high-affinity.Modification of the BoNT/B-H_(C) protein sequence can be performed byeither targeted mutagenesis (site-directed mutagenesis) or randommutagenesis of each amino acid residue within the region known forbinding Syt I/II. These Syt binding regions are well defined by previousco-crystal structural studies^(25,26). For instance, it composes ofresidues 1078-1291 in BoNT/B1 sequence (GenBank access No.: P10844).Although these studies were performed using BoNT/B1 sequence, allsubtypes of BoNT/B can be used as the template to re-create the same orsimilar mutations as described here. Although the exact position forselected residues may not be identical in different BoNT/B subtypes, theanalogous residues in the different BoNT/B subtypes can be easilyidentified (e.g., by sequence alignment when not at exactly the sameposition).

Specifically studied were all residues in the Syt II binding interfaceof BoNT/B based on reported BoNT/B-Syt II complex structure (PDB ID:2NM1), including K1113, D1115, S1116, P1117, V1118, W1178, Y1181, Y1183,E1191, K1192, F1194, A1196, P1197, S1199, S1201, E1203, F1204, E1245,and Y1256, as listed in FIG. 3B.

Mutagenesis is a common laboratory technique to create engineeredprotein products. Several methods to introduce mutations, includingsite-directed mutagenesis, random mutagenesis, combinatorialmutagenesis, and insertional mutagenesis, are available. Site-directedmutagenesis and random mutagenesis were applied to introduce mutationsinto BoNT/B-H_(C) sequence. Random mutagenesis is a powerful tool tocreate mutant libraries for screening. By using PCR primers with dopednucleotides, a pool of mutants was created containing substitutions toall other 19 amino acids at the positions listed in FIG. 3B. Thescreening was then performed using BACTH system as illustrated in FIG.4.

Residues at position 1191, 1178, 1183, and 1199 were identified asimportant for substitution mutations to generate BoNT/B-H_(C) mutantswith enhanced binding to human Syt II. The specific substitutions wereE1191M/C/V/Q/L/Y, Y1183C/P, S1199Y/W/E/H, W1178Y/Q/A/S (FIG. 5A, B).

Further quantitative assays revealed that changing residue E1991 toM/C/V/Q resulted in the strongest binding to h-Syt II, as measured byβ-galactosidase activity (FIG. 6A) and pull-down assays (FIG. 6B).Therefore, E1191 was identified as the primary site to introducemutations that enhance binding to h-Syt II, and substitution mutationsof E1191/M/C/V/Q were identified as the primary mutations.

E1191M was used as the primary mutation to explore whether adding asecondary mutation can further enhance binding to h-Syt II (FIG. 7A).Because Y1183C/P, S1199Y/W/E/H, and W1178Y/Q/A/S substitution mutationsresulted in “light blue” colonies in BACTH screening (FIG. 5B), thesesites were selected as the potential secondary mutation site. Doublemutations combining E1191M with these potential secondary mutations weregenerated and tested. Three mutations, S1199W, S1199Y, and W1178Q werefound to enhance binding to h-Syt II, when combined with the primarymutation at E1191M in pull-down assays (FIG. 7A, B). These threesubstitution mutations were selected as secondary mutations.

Further quantitative assays were carried out to determine the bindingaffinity between BoNT/B-H_(C) mutants and h-Syt II, using bio-layerinterferometry assay as described in FIG. 8A. Double mutations, with onefrom primary mutations (M1191M/C/Q/V) and the other from secondarymutations (S1199W/Y, W1178Q), were measured for their binding affinitiesto h-Syt II (FIG. 8B). The results showed that the following doublemutations: E1191M/C/Q/V combined with S1199W/Y, and E1191M/C/V combinedwith W1178Q have significantly enhanced binding affinities for h-Syt II(FIG. 8B). These eleven double combinations were thus identified asmutations in BoNT/B-H_(C) that enhance binding to h-Syt II the most.

Engineered BoNT/B-H_(C) mutants were found not only enhance binding tohuman Syt II, but also to human Syt I. E1191M/S1199Y and E1191V/S1199Ywere used to show that these mutants displayed significantly enhancedbinding abilities to human Syt I as well as compared to WT BoNT/B-H_(C)(FIG. 9).

The modified BoNT/B-H_(C) mutants may contain amino acids substitutionsat one or combinations of the amino acid residues E1191, Y1183, W1178and S1199 such as E1191M/S1199W, E1191M/S1199Y, E1191M/W1178Q,E1191C/S1199W, E1191C/S1199Y, E1191C/W1178Q, E1191Q/S1199W,E1191Q/S1199Y, E1191V/S1199W, E1191V/S1199Y, and E1191V/W1178Q.

Selected triple mutations by combining mutations at E1191/S1199/W1178sites were also tested and found to have similar binding affinity as thedouble mutation at E1191/S1199 sites. For instance, E1191M/S1199W/W1178Qhas a similar binding affinity as E1191M/S1199W (FIG. 8B). Therefore,triple mutations exhibited the enhanced binding affinity of the doublemutations, although they did not seem to offer significant advantageover double mutations with respect to increased binding affinity.

These results indicate that polypeptides containing BoNT/B-H_(C) withmodified amino acid sequence related to the sequence of WT BoNT/B-H_(C),wherein the modified BoNT/B-H_(C) has improved ability to bind human SytI and II as compared to WT BoNT/B-H_(C) can be made and usedtherapeutically. The polypeptides may be, for example, in the form offull-length BoNT/B mutants, truncated BoNT/B mutants or recombinedproteins that contain the same amino acid substitutions within thereceptor binding domain (BoNT/B-H_(C)), as described above, and also,BoNT/B subtypes with amino acid substitutions at according positions. Inaddition, BoNT/B subtype can be modified to bind h-Syt II by replacingthe residues within their receptor binding domain that are differentfrom BoNT/B1.

The full-length BoNT/B mutants encompassed contain amino acidssubstitutions at one or combinations (e.g., 2 or 3) of the amino acidresidues E1191, Y1183, W1178 and S1199 such as E1191M/S1199W,E1191M/S1199Y, E1191M/W1178Q, E1191C/S1199W, E1191C/S1199Y,E1191C/W1178Q, E1191Q/S1199W, E1191Q/S1199Y, E1191V/S1199W,E1191V/S1199Y, and E1191V/W1178Q. The mutations can be made in the samemanner as disclosed above for BoNT/B-H_(C), using any one of BoNT/Bsubtypes as templates. These mutated BoNT/B toxins gain enhanced bindingto both human Syt II and human Syt I, therefore will achieve higherefficacy and specificity to target human neurons than WT BoNT/B.

REFERENCES FOR THE EXAMPLES SECTION

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The invention claimed is:
 1. A polypeptide comprising a modifiedreceptor binding domain of Clostridial botulinum serotype B (B-H_(c)),wherein the modified B-H_(c) comprises two or more amino acidsubstitutions relative to a reference wild type receptor binding domainof a Clostridial botulinum serotype B, strain 1 as found in SEQ ID NO:5, wherein the two or more amino acid substitutions are at positionsselected from the group consisting of E1191, S1199, W1178, Y1183, andcombinations thereof, wherein: substitution at position 1191 is E1191A;substitution at position 1199 is selected from the group consisting ofS1199W, S1199E, and S1199H; substitution at position 1178 is selectedfrom the group consisting of W1178Y, W1178Q, W1178A, and W1178S; andsubstitution at position 1183 is selected from the group consisting ofY1183C, and Y1183P, wherein the polypeptide is characterized by enhancedbinding to human Syt II as compared to that observed with the referencewild type receptor binding domain.
 2. The polypeptide of claim 1,wherein: i) the modified B-H_(c) possesses 95% or more amino acidsequence identity to the reference wild type receptor binding domain;ii) the modified B-H_(c) corresponds to residues 1078-1291 of SEQ ID NO:5 or residues 859-1291 of SEQ ID NO: 5; iii) the modified B-H_(c) is ofstrain 1; or iv) a combination thereof.
 3. The polypeptide of claim 1that is a botulinum neurotoxin (BoNT) polypeptide comprising: a) aprotease domain; b) a protease cleavage site; c) a translocation domain;and d) the modified B-H_(c).
 4. The polypeptide of claim 3, wherein theprotease domain, translocation domain, and protease cleavage site arefrom serotype selected from the group consisting of A, B, C, D, E, F, G,and combinations thereof.
 5. The polypeptide of claim 1, wherein oneamino acid substitution is W1178Y, W1178A, or W1178S, and thepolypeptide further includes a E1191M substitution.
 6. The polypeptideof claim 1, wherein the modified B-He comprises: i) three substitutionmutations; or ii) three substitution mutations wherein the threesubstitution mutations are at positions that correspond to E1191, Y1183and S1199 or to E1191, S1199 and W1178.
 7. A chimeric moleculecomprising a first portion that is a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)) linked to a second portion,wherein the modified B-H_(c) comprises two or more amino acidsubstitutions relative to a reference wild type receptor binding domainof a Clostridial botulinum serotype B, strain 1 as found in SEQ ID NO:5,wherein the two or more amino acid substitutions are at positionsselected from the group consisting of E1191, S1199, W1178, Y1183, andcombinations thereof, wherein: substitution at position 1191 is E1191A;substitution at position 1199 is selected from the group consisting ofS1199W, S1199E, and S1199H; substitution at position 1178 is selectedfrom the group consisting of W1178Y, W1178Q, W1178A, and W1178S; andsubstitution at position 1183 is selected from the group consisting ofY1183C, and Y1183P, wherein the chimeric molecule is characterized byenhanced binding to human Syt II as compared to that observed with thereference wild type receptor binding domain.
 8. The chimeric molecule ofclaim 7, wherein the first portion and the second portion are linkedcovalently or are linked non-covalently, and wherein the second portionis selected from the group consisting of a small molecule, a nucleicacid, a short polypeptide and a protein.
 9. A pharmaceutical compositioncomprising the polypeptide of claim
 1. 10. A kit comprising apharmaceutical composition of claim 9, and directions for therapeuticadministration of the pharmaceutical composition.
 11. The polypeptide ofclaim 4, wherein the protease domain, translocation domain, and proteasecleavage site are from serotype B, strain 1 or from serotype A,strain
 1. 12. The polypeptide of claim 6, wherein the three substitutionmutations correspond to E1191M, S1199W and W1178Q.
 13. The chimericmolecule of claim 8, wherein the second portion is a bioactive molecule,a therapeutic polypeptide or a non-polypeptide drug.
 14. Thepharmaceutical composition of claim 9, wherein the pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient.